Dave's+Notebook

include component="page" wikiName="genefish" page="Dave's Notebook 2" editable="1"
 * September 13, 2011**

Fed clams and oysters at 4:00pm. spreadsheet

Worked up qPCR data a bit more. Have not done statistics yet. Concerned that actin may be influenced by OA. See paper. Will need to test this and if actin changes with treatments will need to normalize to RNA for now...hopefully NGS will dig up some valid housekeeping genes.


 * September 12, 2011**

FINALLY finished processing images of clam larvae from OA experiment for size analysis. Excel sheet is here. Graphically summary of size analysis +/- SE

Submitted qPCR data to pcrminer for analysis.

Worked more on measure clam larvae using ImageJ
 * September 11, 2011**

Set up qPCR for actin, GPx, and Thioredoxin

Clam OA seed experiment
 * September 9, 2011**

Emma and I drove to Ballard to pick up algae and juvenile clams for the preliminary HS/OA trial. Oyster's were put at 18C to acclimate for 2 weeks (until we get back from PCSGA).

Clams (and oysters) will be fed twice daily at a concentration of 60,000 cells/ml At the moment, we are going to try draining the round tank that they are being held in into another reservoir and then add back a known volume with algae. Once algae is cleared, the water that was pumped into the reservoir will be pumped back into the round tank....this should reduce the amount of algae we burn through as well as minimize seawater waste.

Extracted RNA from OA clams for NGS using tri reagent. Extracted samples consisted of whole jars of larvae (~30,000). Samples were resuspended in 90ul of water. Tons of RNA! Pooled equal quantities (20ug) of 1C3/1C4 and 4C1/4C3. Need 100ng mRNA at the VERY least. Assuming 1% recovery of mRNA from total RNA that means at the very least I should send 10ug. To be very conservative I'm shooting for a total RNA value of 40ug RNA/treatment. This should yield 400ng mRNA 1C3: 13ul (1000ppm) 1C4: 25.5ul (1000ppm) 4C1: 22ul (400ppm) 4C3: 25ul (400ppm) Spec samples again to determine final concentration of pooled samples to send for sequencing. Total RNA for each sample = 40ug!
 * September 7, 2011**
 * Sample ID || User ID || Date || Time || ng/ul || A260 || A280 || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || 340 raw ||
 * 1C3 || Default || 9/7/11 || 1:25 PM || 1533.97 || 38.349 || 19.014 || 2.02 || 1.17 || 40 || 230 || 32.847 || 3.625 ||
 * 1C4 || Default || 9/7/11 || 1:26 PM || 783.22 || 19.58 || 9.288 || 2.11 || 1.92 || 40 || 230 || 10.212 || 0.475 ||
 * 4C1 || Default || 9/7/11 || 1:26 PM || 913.96 || 22.849 || 11.638 || 1.96 || 1.02 || 40 || 230 || 22.505 || 3.874 ||
 * 4C3 || Default || 9/7/11 || 1:27 PM || 791.61 || 19.79 || 9.536 || 2.08 || 1.48 || 40 || 230 || 13.385 || 1.403 ||
 * Sample ID || User ID || Date || Time || ng/ul || A260 || A280 || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || 340 raw ||
 * 400ppm || Default || 9/7/11 || 4:01 PM || 848.93 || 21.223 || 10.514 || 2.02 || 1.21 || 40 || 230 || 17.471 || 2.128 ||
 * 1000ppm || Default || 9/7/11 || 4:02 PM || 1099.54 || 27.489 || 13.433 || 2.05 || 1.42 || 40 || 230 || 19.414 || 1.385 ||

The primers arrived!!
 * August 25, 2011**

Set up qPCR assays for ALL primer pairs. Ran all samples in duplicate. Used PCR miner to calculate PCR efficiency. Normalized to beta-actin.

DNase treated RNA samples from August 22nd, 2011 Final volume of samples = 50ul
 * August 23, 2011**

Set up RT reaction as follows


 * Sample ID || User ID || Date || Time || ng/ul || .5ug ||
 * Day 0 DNase || Default || 8/23/11 || 11:42 AM || 216.99 || 2.304253652 ||
 * 1C1 7/29 DNase || Default || 8/23/11 || 11:42 AM || 34.53 || 14.48016218 ||
 * 1C4 8/5 DNase || Default || 8/23/11 || 11:43 AM || 18.48 || 27.05627706 ||
 * 4C3 7/29 DNase || Default || 8/23/11 || 11:43 AM || 34.05 || 14.68428781 ||
 * 4C2 8/5 DNase || Default || 8/23/11 || 11:44 AM || 12.78 || 39.12363067 ||
 * buffer || 5 ||  ||   ||   ||   ||
 * dntp || 5 ||  ||   ||   ||   ||
 * RT || 1 ||  ||   ||   ||   ||
 * water || 0 ||  ||   ||   ||   ||
 * RT || 1 ||  ||   ||   ||   ||
 * water || 0 ||  ||   ||   ||   ||

Extracted RNA from clam larvae sampled on 7/29 and 8/5 using TriReagent. Only exctracted one sample from each date of the 400ppm and 1000ppm treatment as a preliminary test to see if A) I can get enough RNA to do qPCR, and B) if the qPCR assays actually work.
 * August 22, 2011**

Notes: Spun samples at 1500 x g for 1min at 4C to collect larvae at bottom of tube before removing sea water and adding TriReagent.

Jar pH for Clam OA experiment Trial1
 * August 19, 2011**

Clam OA trail1 percent survival by jar Numbers represent percent survival by individual jars of the initial total count.

Below is another way of looking at changing densities.

To generate these graphs I subtracted the total larval counts for each jar from the previous sampling date to get an overall change in larval abundance. Initial thought is that it looks kind of messy but maybe that's just the data and not the presentation format.

Since jars were sampled on different dates along the way, what I would hope to see are lines that overlap when sampled on the same day or lines that are parallel (such as 4c1 and 4c6 in the 400 treatment, 1C3 and 1C6 in the 1000 treatment, or 3C3, 3C4, 3C5, and 3C6...possibly 3C1 as well...in the 520 treatment.)



Next, I took the numbers generated above and expressed them as a percent change compared to the previously recorded total values. My thought was that this should allow for a more direct comparison between jars and eliminate the variability of initial densities or differences mortalities between jars.



Entered mortality data from Clam OA trial into excel.
 * August 18, 2011**

Below is a graph of survivorship for the three treatments. To create the graph I first averaged the total counts and total live counts for the individual jar replicates to come up with a single number for each jar. I then averaged the total counts and total jar counts for each jar from each treatment to give me a single value for each treatment on each sampling day. I then calculated the percent survival by dividing the total live/total counts for each treatment. Below is are "density" graphs for each jar from each treatment. To generate these numbers I divided the total counts for each jar and divided by the total volume for each aliquot to come up with a density/ul and then multiplied by 4500 (the ul that each jar was resuspended in to sample).

Note: These are the TOTAL densities and includes by live and dead larvae.

Clam OA sampling
 * July 29, 2011**

Took genomics samples (~1500) larvae, 2 wells of 3x 25ul aliquots for counts (~50/well) from 1C1, 1C3, 1C4, 3C1, 3C3, 3C4, 4C1, 4C3, and 4C4

Spec pH was done on jars 1C1, 3C1, and 4C1.


 * July 28, 2011**

Picked up algae from nate

2 species...Iso and ?

Algae was taken to NOAA where it was split, counted, and hooked up to feeders. (need to contact Sarah for more detailed feeding protocol)

1st day of clam OA experiment.
 * July 27, 2011**

Manilla clam larvae arrived today from Hawaii. Were shipped priority overnight.

Clams were spawned fertilized July 21st, 2011

Total estimated larvae shipped = 1 million, 5 day old larvae

Larvae were rinsed from mesh into 250ml seawater pCO2 = 400ppm.

Total estimated recovered larvae based on counts = 875,000

Density of larvae in 250ml = 3,500/ml

Aliquot 14ml to each jar (two 7ml aliquots were distributed) = 48,600 larvae/jar.... or ~45,000 as a ballpark estimate.

3 samples of 5000 larvae each were taken for genomics. -samples were left in seawater and snap frozen in LN2

3 samples of 5000 larvae each were taken for genetics. -samples were left in seawater and snap frozen in LN2

July 22, 2011 Exciting find of the day...

MyD88!

Awhile ago when I first started playing with DAVID, my initial observations were just by scrolling though the data and it looked like the NFkB/Toll receptor pathway might be something of interest. In particular was the identification of MyD88.

Upon further analysis of enriched GO terms and their associated genes, these initial observations appear to be confirmed! In addition, the annotated contrig from the RNAseq data contains part of the MyD88 death domain....check it out! MyD88 sequence alignments are to human, mouse, and tongue sole.

Other players that appear in this pathway and were also identified by DAVID are NFkB p105, TRAF3, Tradd...

July 19, 2011 Generating figures for enriched GO terms using DAVID outputs in REViGO

In DAVID -Imported swissprot IDs for BARNS values with 2 fold or higher over MASH, P <0.05, and E-values __<__0.01 -Converted list of swissport IDs to DAVID -Selected "gene ontology" from list of annotation summary results (directions can be found [|here]) -Selected and saved .txt files for individual GO_Fat terms. Note: Also ran Bonferroni and Fold change under "options"

In REViGO -open .txt in excel and isolate GO ID's using "text to columns" function under data tab. Isolate GO ID by designating "~" as the delimiter. - Copy and paste GO terms and p-values from DAVID into REViGO looks something like this... % GO ID p Value GO:00134 0.00821 etc.. etc..

-Run REViGO - Selected "log10 pvalue" so that size of circle illustrates significance (larger is better) - Displayed all terms and rearranged to so they do not overlap -Also displayed interactive report and moved circles so they fit into one field of view.

Summary of output for all GO categories:

And here's a remake of the biological process pie chart in Excel instead of SPSS so that it's actually legible. -Duplicate GO terms were removed as well as all "other etc." GO terms. - Includes all contigs with an E-value__<__0.01

July 12, 2011 Back to the basics.

Working on wrapping up summary of basic NGS results....# reads, coverage, etc. for basic transcriptome characterization.

July 8, 2011 Working my way thought cytoscape

Managed to upload a test file and complete the PiNGO tutorial to identify unannotated candidate genes associated with you biological process of interest

Output looks something like this...

Now if I can just get my RNAseq data to upload to cytoscape...

July 6, 2011 More RNAseq

Playing around with [|REViGO], a GO ontology visualization tool.

Also playing around with Bio4j but havent had much luck. Cant get past GO frequencies to construct visualization

Both appear to have a limit for maximum number of entries. For REViGO I could only submit >2fold changes in Barnstable.

July 5, 2011 Playing around with DAVID Bioinformatic Database

Submitted list of SP ID genes significantly "annotated" (e<.01) and differentially regulated by 2 fold or more to database.

Ran "Functional Annotation Clustering" with high stringency and received an ouput of 29 different gene clusters. 94 submissions were not clustered. .txt summary of output can be found [|here]. Green boxes designate shared pathways. In online output, you can scroll over individual boxes to see which pathways they are associated with.

You can also click on the individual pathway which directs you to a graphical representation of the pathway. The positions of genes submitted in my list are highlighted by red stars. Ex: NfKb pathway. Could also have done Toll-like receptor signaling among others.

Wanted to identify other members of the above pathway that are not transcriptionally regulated.

Submitted new list of SPID that contain all significantly annotated genes. Could not include other identifiers because the addition of un annotated or genes not significant exceeds the 2000 gene limit of DAVID.

Found a few more!

Compiled all piecharts and scatterplots into single ppt. doc that can be found [|here].
 * June 17, 2011**
 * More RNAseq analysis**

Two quick observations: 1. ~50% of the contigs are unannotated 2. There appears to be a lot more happening in the barnstable clams, however, it's hard to say for sure since so many of the contigs are not annotated.

Need to go through what some of the more interesting GO terms are and see what the individual gene are all about.

Only thing still left in limbo is installing the freshly cleaned filters, filling resevoir with water for bubbling, and removing jars from freshwater soak. Nothing will really happen with these loose ends until I receive confirmation that we have larvae on the way since the filters should be stored dried, I have no way of storing bubbled water and that would be a waste of gas, and there's no harm in soaking the jars longer.
 * June 16, 2011**
 * Finished plumbing gas lines for designer gas system in the basement**.

Not sure the best way to keep track of this type of data analysis so I'll try just listing off what I do and linking every file I make along the way. Note: All files were generated independent of what Steven has done so the Contig#'s and completely different!
 * Clam RNAseq analysis (more piecharts and scatterplots)!**

1. Ran RNAseq experiment with Barnstable as 1st variable and Mashpee as second variable. Assuming this translates as Barnstable vs. Mashpee

2. Combined RNAseq experiment file with the following 3 GO files in Galaxy to generate Go annotation file.

3. Removed duplicate Contig/Go_slim terms [|here].

4. Removed all non significant contigs [|here].

5. Next I divided the dataset according to GO Category and made separate file that contained significant contigs only. [|Cell Function] [|Cell Function Significant Contigs only] [|Cell Process] [|Cell Process Significant Contigs only] [|Cell Component] [|Cell Component Significant Contigs only]

All files contain column that further divides contigs into the following categories: Not Annotated (E-value >0.01) Not Significant (P>0.05) Significant 2 Fold Change (+ or -) (2__<__x<4) 4 Fold Change (+ or -) (x__>__4) For clarification, designations are listed in ascending order of specificity. In otherwords, everything not designated as "not annotated" is annotated, everything"not significant" is significant, things considered significant are not 2 or 4 fold, all 2 or 4 fold changes are significant.

5. Generated data files with increased stringency based on GO category. [|Cell Function Significant and Annotated] [|Cell Function Significant, Annotated, and 2 Fold or Higher] [|Cell Process Significant and Annotated] [|Cell Process Significant, Annotated, and 2 Fold or Higher] [|Cell Component Significant and Annotated] [|Cell Component Significant, Annotated, and 2 Fold or Higher]

In order to generate pie charts by GO_Slim term I had to generate files in which all contigs that were not considered annotated has "not annotated" in place of their GO_slim term. [|Cell Function with not annotated replacement] [|Cell Process with not annotated replacement] [|Cell Component with not annotated replacement]

6. Now going to generate logs of pie charts and scatter plots to try and tease out some patterns from the data.

Rumor has it that the conditions are prime for abalone spawning at Muk which means I need to crank construction of the system into hight gear.
 * June 15, 2011**
 * Building designer gas system in the basement**

Everything is finished except the following: I tried filling a bin with seawater to prep it for bubbling but when i turned the pump on the water came out smelling like sulfur. I have since rinsed the filters in bortex and they are drying in the basement. I will get that system set up tomorrow morning once the filters have dried. The bins are ready to go, just need to plum in the gas lines which is a few simple cuts of some tubing and we should be all set. Jars are still soaking, I will leave those until tomorrow afternoon at the earliest. If no larvae I'll continue to let them soak.

Waiting for Elene and Brent to replace some part on the other regulator for the gas tanks.

OA system update: I spent the morning driving around town tracking down totes to use for some designer gas experiments. I ended up purchasing [|6 of the following 63L totes from "The Container Store" in bellevue].
 * June 14, 2011**

After messing around in the basement for awhile, I managed to fit 6 of the 4.5L pretzel beakers into one of these totes. 4.5L jars are the same as those used at the NOAA facility. Six of these jars is the maximum that can fit the length of the totes, but there is still ~1in of space in the height, so it is possible to fit a larger jar but this would require a bit of searching around.

From an experimental design standpoint, we would need to set up two totes minimum for each pCO2 treatment. This would provide a total of 54L for each treatment. If we pack 2,000 larvae/liter that would give us 12,000 larvae/jar and 108,000 total larvae/ treatment.

More M.merc qPCR graphs Because the relative expression values for graphs generated on June 10, 2011 look very suspicious (everything is down in Vt and too many values are identical to Actin....just doesnt seem right), I have gone back and used the generic equation to calculate relative expression values and normalized to Actin.
 * June 13, 2011**

Note: I had to break the assays into multiple graphs because the scale of some dwarfed the others...

Results from M.merc qPCR primer test June 9, 2011 Relative expression values were calculated using deltaCq method in CFX manager software followed by normalization to Actin.
 * June 10, 2011**

Testing primers for M.merc that appear to work based initial screening done by both Sam and me ( June 3, 2011).
 * June 9, 2011**

Pooled equal volumes of cDNA to create the following 4 pools of cDNA templates. BX (contains all samples from June and August) MA (contains all samples from June and August) Vt control (contains all control samples from Vt exposure exp) Vt (contains all samples from challenge group of Vt exposure exp)

qPCR data file and plate map can be found [|here], [|here], and [|here]. 1st link = full plate 2nd link = 2nd plate with remaining primers 3rd link = actin and serine protease inhibitor because I forgot to add them on the 2nd plate.

Testing primers from M.merc/QPX analysis Used 2ul pooled template cDNA from May 26, 2011
 * June 3, 2011**

Plate map: Names correspond to primer pair used. Results: Overall the test looks really good Primers that did not amplify: AMPN
 * || 1 || 2 || 3 || 4 ||
 * A || MPEG || MPEG || NERM || NERM ||
 * B || AMPN || AMPN || DEF1 || DEF1 ||
 * C || LEC2 || LEC2 || SAMH1 || SAMH1 ||
 * D || NEP || NEP || FCN-1 || FCN-1 ||
 * E || APLY || APLY || PXDN || PXDN ||
 * F || OXLA || OXLA || NTC || NTC ||
 * G || IF44L || IF44L || NTC || NTC ||
 * H || CO2 || CO2 || NTC || NTC ||

Primers with multiple/fat melting curve peaks: APLY OXLA SAMH1? (is probably ok)

Ct was <30 ("high expression") DEF1 LEC2

Ct was 30-35 MPEG NEP PXDN CO2

Ct was >35 NERM SAMH1 FCN-1 IF44L Primers designed on May 12, 2011 were reconstituted at 100uM and filed in the primer stocks log Box#9 Made cDNA from DNased M.merc RNA for the QPX project for future use to test genes identified from NGS analysis.
 * May 26, 2011**

RT Rxn was carried out in a 96 well plate to make future qPCR template loading easier. Plate map of cDNA is as follows.
 * || 5 || 6 || 7 || 8 || 9 ||
 * A || BX 1-3 || MA 1-3 || BX 1-1 || MA 1-1 || BX 3-2 ||
 * B || BX 1-4 || MA 1-5 || BX 1-2 || MA 1-4 || MA 1-4 ||
 * C || BX 2-2 || MA 2-4 || BX 2-1 || MA 2-1 || BX 4-1 ||
 * D || BX 2-3 || MA 2-5 || BX 2-2 || MA 2-2 || BX 4-2 ||
 * E || BX 3-2 || MA 3-3 || BX 3-2 || MA 3-1 || MA 4-1 ||
 * F || BX 3-3 || MA 3-4 || BX 3-3 || MA 3-2 || MA 4-2 ||
 * G || BX 4-1 || MA 4-3 || BX 4-1 || MA 4-1 || NTC ||
 * H || BX 4-2 || MA 4-4 || BX 4-2 || MA 4-2 || NTC ||
 * |||| 6/24/10 |||| 9/2/10 ||  ||

Notes: A9 & B9 were pooled and diluted 1:2 to be used as template from primer testing C9, D9, E9, & F9 will be pooled and used to make the standard curve.

My first pie chart...



DNased hard RNA samples from March, 17 2011 (these are the same samples used to make the libraries) Note: Not enough of MA 1-2 or BX 3-1 samples remained after pooling for library so I extracted RNA from MA 1-4 and BX 3-3 to replace.
 * May 18, 2011**

DNased hard clam RNA samples extracted on May 13, 2011
 * May 17, 2011 **
 * Sample ID || User ID || Date || Time || ng/ul || A260 || A280 || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || 340 raw ||
 * DNased CA 1 || Default || 5/17/2011 || 2:19 PM || 148.6 || 3.715 || 1.872 || 1.98 || 1.59 || 40 || 230 || 2.332 || -0.021 ||
 * DNased CA 2 || Default || 5/17/2011 || 2:20 PM || 203.35 || 5.084 || 2.583 || 1.97 || 1.64 || 40 || 230 || 3.094 || 0.016 ||
 * DNased CA 3 || Default || 5/17/2011 || 2:20 PM || 150.92 || 3.773 || 1.902 || 1.98 || 1.62 || 40 || 230 || 2.326 || -0.005 ||
 * DNased CA 4 || Default || 5/17/2011 || 2:21 PM || 170.06 || 4.252 || 2.138 || 1.99 || 1.42 || 40 || 230 || 2.999 || -0.009 ||
 * DNased CA 5 || Default || 5/17/2011 || 2:21 PM || 168.13 || 4.203 || 2.111 || 1.99 || 1.63 || 40 || 230 || 2.58 || -0.004 ||
 * DNased CA 6 || Default || 5/17/2011 || 2:22 PM || 173.49 || 4.337 || 2.215 || 1.96 || 1.45 || 40 || 230 || 3.001 || -0.019 ||
 * DNased CA 7 || Default || 5/17/2011 || 2:22 PM || 147.72 || 3.693 || 1.888 || 1.96 || 1.51 || 40 || 230 || 2.441 || -0.001 ||
 * DNased CA 8 || Default || 5/17/2011 || 2:23 PM || 113.92 || 2.848 || 1.491 || 1.91 || 1.16 || 40 || 230 || 2.453 || -0.025 ||
 * DNased MA 1 || Default || 5/17/2011 || 2:23 PM || 143.83 || 3.596 || 1.808 || 1.99 || 1.59 || 40 || 230 || 2.261 || -0.003 ||
 * DNased MA 2 || Default || 5/17/2011 || 2:24 PM || 154.62 || 3.865 || 1.93 || 2 || 1.67 || 40 || 230 || 2.319 || -0.009 ||
 * DNased MA 3 || Default || 5/17/2011 || 2:24 PM || 64.12 || 1.603 || 0.846 || 1.89 || 0.92 || 40 || 230 || 1.739 || 0.108 ||
 * DNased MA 4 || Default || 5/17/2011 || 2:25 PM || 153.87 || 3.847 || 1.954 || 1.97 || 1.26 || 40 || 230 || 3.063 || -0.017 ||
 * DNased MA 5 || Default || 5/17/2011 || 2:25 PM || 156.09 || 3.902 || 1.99 || 1.96 || 1.47 || 40 || 230 || 2.648 || -0.007 ||
 * DNased MA 6 || Default || 5/17/2011 || 2:25 PM || 110.01 || 2.75 || 1.437 || 1.91 || 1.06 || 40 || 230 || 2.598 || 0.26 ||
 * DNased MA 7 || Default || 5/17/2011 || 2:26 PM || 152.52 || 3.813 || 1.929 || 1.98 || 1.57 || 40 || 230 || 2.432 || -0.015 ||
 * DNased MA 8 || Default || 5/17/2011 || 2:26 PM || 178.88 || 4.472 || 2.324 || 1.92 || 1.05 || 40 || 230 || 4.256 || -0.017 ||
 * DNased MAX 1 || Default || 5/17/2011 || 2:27 PM || 146.63 || 3.666 || 1.893 || 1.94 || 1.53 || 40 || 230 || 2.398 || 0.001 ||
 * DNased MAX 2 || Default || 5/17/2011 || 2:27 PM || 168.77 || 4.219 || 2.154 || 1.96 || 1.68 || 40 || 230 || 2.513 || 0.013 ||
 * DNased MAX 3 || Default || 5/17/2011 || 2:28 PM || 188.57 || 4.714 || 2.28 || 2.07 || 1.73 || 40 || 230 || 2.733 || -0.001 ||
 * DNased MAX 4 || Default || 5/17/2011 || 2:28 PM || 173.86 || 4.346 || 2.141 || 2.03 || 1.67 || 40 || 230 || 2.601 || -0.007 ||
 * DNased MAX 5 || Default || 5/17/2011 || 2:29 PM || 160.75 || 4.019 || 2.045 || 1.97 || 1.59 || 40 || 230 || 2.52 || 0.003 ||
 * DNased MAX 6 || Default || 5/17/2011 || 2:29 PM || 149.21 || 3.73 || 1.893 || 1.97 || 1.32 || 40 || 230 || 2.828 || 0.043 ||
 * DNased MAX 7 || Default || 5/17/2011 || 2:30 PM || 157.32 || 3.933 || 1.963 || 2 || 1.62 || 40 || 230 || 2.423 || -0.002 ||
 * DNased MAX 8 || Default || 5/17/2011 || 2:30 PM || 206.76 || 5.169 || 2.594 || 1.99 || 1.55 || 40 || 230 || 3.339 || -0.003 ||

Exctracted RNA from hard clam gill tissue Rec'd from NewJersey 6/24/2010
 * May 13, 2011 **
 * Sample ID || User ID || Date || Time || ng/ul || A260 || A280 || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || 340 raw ||
 * CA 1 || Default || 5/13/11 || 2:00 PM || 744.68 || 18.617 || 9.619 || 1.94 || 2.09 || 40 || 230 || 8.899 || -0.008 ||
 * CA 2 || Default || 5/13/11 || 2:01 PM || 1279.75 || 31.994 || 16.205 || 1.97 || 1.93 || 40 || 230 || 16.584 || 0.217 ||
 * CA 3 || Default || 5/13/11 || 2:01 PM || 1494.8 || 37.37 || 18.918 || 1.98 || 2.12 || 40 || 230 || 17.614 || 0.05 ||
 * CA 4 || Default || 5/13/11 || 2:02 PM || 708.33 || 17.708 || 9.026 || 1.96 || 1.79 || 40 || 230 || 9.88 || 0.055 ||
 * CA 5 || Default || 5/13/11 || 2:02 PM || 872.5 || 21.812 || 11.188 || 1.95 || 2.05 || 40 || 230 || 10.657 || 0.049 ||
 * CA 6 || Default || 5/13/11 || 2:03 PM || 909.03 || 22.726 || 11.609 || 1.96 || 1.77 || 40 || 230 || 12.821 || 0.027 ||
 * CA 7 || Default || 5/13/11 || 2:03 PM || 1015.16 || 25.379 || 12.887 || 1.97 || 1.99 || 40 || 230 || 12.773 || 0.207 ||
 * CA 8 || Default || 5/13/11 || 2:04 PM || 1935.36 || 48.384 || 24.691 || 1.96 || 1.65 || 40 || 230 || 29.263 || 0.293 ||
 * MA 1 || Default || 5/13/11 || 2:04 PM || 1073.21 || 26.83 || 13.57 || 1.98 || 2.04 || 40 || 230 || 13.154 || 0.106 ||
 * MA 2 || Default || 5/13/11 || 2:05 PM || 1216.03 || 30.401 || 15.245 || 1.99 || 2.12 || 40 || 230 || 14.343 || 0.125 ||
 * MA 3 || Default || 5/13/11 || 2:05 PM || 131.84 || 3.296 || 1.811 || 1.82 || 1.31 || 40 || 230 || 2.526 || 0.264 ||
 * MA 4 || Default || 5/13/11 || 2:06 PM || 563.15 || 14.079 || 7.283 || 1.93 || 1.54 || 40 || 230 || 9.149 || 0.062 ||
 * MA 5 || Default || 5/13/11 || 2:06 PM || 589.22 || 14.73 || 7.606 || 1.94 || 1.84 || 40 || 230 || 8.011 || 0.043 ||
 * MA 6 || Default || 5/13/11 || 2:07 PM || 230.71 || 5.768 || 3.176 || 1.82 || 1.36 || 40 || 230 || 4.245 || 0.691 ||
 * MA 7 || Default || 5/13/11 || 2:07 PM || 636.32 || 15.908 || 8.202 || 1.94 || 2.04 || 40 || 230 || 7.807 || 0.049 ||
 * MA 8 || Default || 5/13/11 || 2:08 PM || 808.22 || 20.206 || 10.4 || 1.94 || 1.13 || 40 || 230 || 17.907 || 0.081 ||
 * MAX 1 || Default || 5/13/11 || 2:08 PM || 1532.95 || 38.324 || 19.456 || 1.97 || 2.03 || 40 || 230 || 18.851 || 0.224 ||
 * MAX 2 || Default || 5/13/11 || 2:09 PM || 1608.46 || 40.212 || 20.41 || 1.97 || 2.12 || 40 || 230 || 18.98 || 0.246 ||
 * MAX 3 || Default || 5/13/11 || 2:09 PM || 925.59 || 23.14 || 11.818 || 1.96 || 2.07 || 40 || 230 || 11.158 || 0.083 ||
 * MAX 4 || Default || 5/13/11 || 2:09 PM || 881.42 || 22.035 || 11.394 || 1.93 || 2.1 || 40 || 230 || 10.479 || 0.131 ||
 * MAX 5 || Default || 5/13/11 || 2:10 PM || 1096.15 || 27.404 || 14.031 || 1.95 || 1.94 || 40 || 230 || 14.148 || 0.244 ||
 * MAX 6 || Default || 5/13/11 || 2:10 PM || 799.57 || 19.989 || 10.229 || 1.95 || 1.59 || 40 || 230 || 12.602 || 0.453 ||
 * MAX 7 || Default || 5/13/11 || 2:11 PM || 894.79 || 22.37 || 11.442 || 1.96 || 2.15 || 40 || 230 || 10.392 || 0.198 ||
 * MAX 8 || Default || 5/13/11 || 2:11 PM || 703.41 || 17.585 || 9.037 || 1.95 || 1.72 || 40 || 230 || 10.253 || 0.139 ||

Ordered qPCR primers for the following hard clam genes. SRID = 1315-1340 SRID for primers by Steven/Sam = 1233-1312 Note: I sorted back through them to eliminate ones that Steven had already identified as well as ones I didnt think would be quite as interesting anymore.
 * May 12, 2011**

Final list
 * FeatureID || Fold Change (original values) || evalue || ID3 ||
 * ConsensusfromContig5346 || 120.76 || 2.00E-10 || AMPN_BOVIN Aminopeptidase N OS=Bos taurus GN=ANPEP PE=2 SV=4 ||
 * ConsensusfromContig8045 || 6.967 || 4.00E-05 || NEURM_APLCA Neuromacin-like protein OS=Aplysia californica PE=3 SV=1 ||
 * ConsensusfromContig5822 || 3.821 || 5.00E-16 || IF44L_MOUSE Interferon-induced protein 44-like OS=Mus musculus GN=IFI44L PE=2 SV=2 ||
 * ConsensusfromContig6406 || 2.528 || 5.00E-04 || NEP_PONAB Neprilysin OS=Pongo abelii GN=MME PE=2 SV=2 ||
 * ConsensusfromContig7255 || 2.218 || 1.00E-08 || APLY_APLKU Aplysianin-A OS=Aplysia kurodai PE=1 SV=1 ||
 * ConsensusfromContig2773 || 2.074 || 5.00E-04 || OXLA_DEMVE L-amino-acid oxidase OS=Demansia vestigiata PE=2 SV=1 ||
 * ConsensusfromContig4978 || 1.901 || 6.00E-20 || TECR_BOVIN Trans-2,3-enoyl-CoA reductase OS=Bos taurus GN=TECR PE=2 SV=1 ||
 * ConsensusfromContig1643 || 1.592 || 4.00E-13 || SAMH1_DANRE SAM domain and HD domain-containing protein 1 OS=Danio rerio GN=samhd1 PE=2 SV=2 ||
 * ConsensusfromContig4820 || 1.542 || 3.00E-04 || DEF1_CRAVI Defensin-1 OS=Crassostrea virginica PE=1 SV=1 ||
 * ConsensusfromContig1178 || 1.51 || 6.00E-07 || LEC2_SISCA C-type lectin isoform 2 OS=Sistrurus catenatus edwardsii PE=2 SV=1 ||
 * ConsensusfromContig3464 || 1.478 || 3.00E-04 || CO2_HUMAN Complement C2 OS=Homo sapiens GN=C2 PE=1 SV=2 ||
 * ConsensusfromContig1063 || -61.814 || 1.00E-05 || MPEG1_BOVIN Macrophage-expressed gene 1 protein OS=Bos taurus GN=MPEG1 PE=2 SV=2 ||
 * ConsensusfromContig5239 || -4.851 || 4.00E-08 || FCN1_RAT Ficolin-1 OS=Rattus norvegicus GN=Fcn1 PE=2 SV=2 ||
 * ConsensusfromContig4755 || -3.101 || 4.00E-09 || PXDN_CAEEL Peroxidasin homolog OS=Caenorhabditis elegans GN=pxn-1 PE=1 SV=1 ||
 * ConsensusfromContig4755 || -3.101 || 4.00E-09 || PXDN_CAEEL Peroxidasin homolog OS=Caenorhabditis elegans GN=pxn-1 PE=1 SV=1 ||

I spent the morning scrolling through the endless lines from the hard clam RNAseq excel sheet for qPCR candidates. Here are the results from my first pass through the data.
 * May 11, 2011**
 * FeatureID || Fold Change (original values) || evalue || ID3 ||
 * ConsensusfromContig5346 || 120.76 || 2.00E-10 || AMPN_BOVIN Aminopeptidase N OS=Bos taurus GN=ANPEP PE=2 SV=4 ||
 * ConsensusfromContig8045 || 6.967 || 4.00E-05 || NEURM_APLCA Neuromacin-like protein OS=Aplysia californica PE=3 SV=1 ||
 * ConsensusfromContig7486 || 6.218 || 1.00E-09 || CYTA_MOUSE Cystatin-A OS=Mus musculus GN=Csta PE=2 SV=1 ||
 * ConsensusfromContig7364 || 4.779 || 1.00E-06 || COL12_CHICK Collectin-12 OS=Gallus gallus GN=COLEC12 PE=2 SV=1 ||
 * ConsensusfromContig5822 || 3.821 || 5.00E-16 || IF44L_MOUSE Interferon-induced protein 44-like OS=Mus musculus GN=IFI44L PE=2 SV=2 ||
 * ConsensusfromContig4839 || 2.973 || 4.00E-11 || LECH_CHICK Hepatic lectin OS=Gallus gallus PE=1 SV=1 ||
 * ConsensusfromContig4446 || 2.679 || 3.00E-20 || DHSO_BOMMO Sorbitol dehydrogenase OS=Bombyx mori GN=SDH PE=2 SV=1 ||
 * ConsensusfromContig6406 || 2.528 || 5.00E-04 || NEP_PONAB Neprilysin OS=Pongo abelii GN=MME PE=2 SV=2 ||
 * ConsensusfromContig983 || 2.366 || 1.00E-09 || RN213_HUMAN RING finger protein 213 OS=Homo sapiens GN=RNF213 PE=1 SV=2 ||
 * ConsensusfromContig8423 || 2.342 || 2.00E-15 || IAP_GVCP Apoptosis inhibitor IAP OS=Cydia pomonella granulosis virus GN=IAP PE=4 SV=1 ||
 * ConsensusfromContig6649 || 2.275 || 3.00E-04 || COX2_HUMAN Cytochrome c oxidase subunit 2 OS=Homo sapiens GN=MT-CO2 PE=1 SV=1 ||
 * ConsensusfromContig6038 || 2.263 || 6.00E-15 || CATL1_CHICK Cathepsin L1 (Fragments) OS=Gallus gallus GN=CTSL1 PE=1 SV=1 ||
 * ConsensusfromContig7255 || 2.218 || 1.00E-08 || APLY_APLKU Aplysianin-A OS=Aplysia kurodai PE=1 SV=1 ||
 * ConsensusfromContig6312 || 2.209 || 2.00E-25 || CATB_BOVIN Cathepsin B OS=Bos taurus GN=CTSB PE=1 SV=5 ||
 * ConsensusfromContig2773 || 2.074 || 5.00E-04 || OXLA_DEMVE L-amino-acid oxidase OS=Demansia vestigiata PE=2 SV=1 ||
 * ConsensusfromContig7192 || 1.989 || 1.00E-41 || PCKG_DROME Phosphoenolpyruvate carboxykinase [GTP] OS=Drosophila melanogaster GN=Pepck PE=2 SV=2 ||
 * ConsensusfromContig4978 || 1.901 || 6.00E-20 || TECR_BOVIN Trans-2,3-enoyl-CoA reductase OS=Bos taurus GN=TECR PE=2 SV=1 ||
 * ConsensusfromContig5245 || 1.769 || 1.00E-08 || MANA_MYTED Mannan endo-1,4-beta-mannosidase OS=Mytilus edulis PE=1 SV=1 ||
 * ConsensusfromContig3453 || 1.673 || 3.00E-07 || DBNL_MOUSE Drebrin-like protein OS=Mus musculus GN=Dbnl PE=1 SV=2 ||
 * ConsensusfromContig1643 || 1.592 || 4.00E-13 || SAMH1_DANRE SAM domain and HD domain-containing protein 1 OS=Danio rerio GN=samhd1 PE=2 SV=2 ||
 * ConsensusfromContig4820 || 1.542 || 3.00E-04 || DEF1_CRAVI Defensin-1 OS=Crassostrea virginica PE=1 SV=1 ||
 * ConsensusfromContig1178 || 1.51 || 6.00E-07 || LEC2_SISCA C-type lectin isoform 2 OS=Sistrurus catenatus edwardsii PE=2 SV=1 ||
 * ConsensusfromContig4480 || 1.496 || 2.00E-08 || IAP1_DROME Apoptosis 1 inhibitor OS=Drosophila melanogaster GN=th PE=1 SV=2 ||
 * ConsensusfromContig3464 || 1.478 || 3.00E-04 || CO2_HUMAN Complement C2 OS=Homo sapiens GN=C2 PE=1 SV=2 ||
 * ConsensusfromContig1063 || -61.814 || 1.00E-05 || MPEG1_BOVIN Macrophage-expressed gene 1 protein OS=Bos taurus GN=MPEG1 PE=2 SV=2 ||
 * ConsensusfromContig2830 || -38.266 || 8.00E-07 || IAP1_DROME Apoptosis 1 inhibitor OS=Drosophila melanogaster GN=th PE=1 SV=2 ||
 * ConsensusfromContig1092 || -18.054 || 2.00E-07 || LOXE3_MOUSE Epidermis-type lipoxygenase 3 OS=Mus musculus GN=Aloxe3 PE=2 SV=1 ||
 * ConsensusfromContig1081 || -12.622 || 5.00E-09 || IAP2_DROME Apoptosis 2 inhibitor OS=Drosophila melanogaster GN=Iap2 PE=2 SV=3 ||
 * ConsensusfromContig4706 || -7.954 || 3.00E-31 || TRAF3_HUMAN TNF receptor-associated factor 3 OS=Homo sapiens GN=TRAF3 PE=1 SV=2 ||
 * ConsensusfromContig5239 || -4.851 || 4.00E-08 || FCN1_RAT Ficolin-1 OS=Rattus norvegicus GN=Fcn1 PE=2 SV=2 ||
 * ConsensusfromContig137 || -3.306 || 2.00E-12 || PGRP_BOMMO Peptidoglycan recognition protein OS=Bombyx mori PE=1 SV=1 ||
 * ConsensusfromContig731 || -3.264 || 3.00E-04 || PXDN_CAEBR Peroxidasin homolog OS=Caenorhabditis briggsae GN=pxn-1 PE=3 SV=1 ||
 * ConsensusfromContig4755 || -3.101 || 4.00E-09 || PXDN_CAEEL Peroxidasin homolog OS=Caenorhabditis elegans GN=pxn-1 PE=1 SV=1 ||
 * ConsensusfromContig4065 || -2.223 || 3.00E-28 || CALX_CANFA Calnexin OS=Canis familiaris GN=CANX PE=1 SV=3 ||
 * ConsensusfromContig7234 || -2.104 || 2.00E-35 || SODC_CHICK Superoxide dismutase [Cu-Zn] OS=Gallus gallus GN=SOD1 PE=1 SV=3 ||
 * ConsensusfromContig7234 || -2.104 || 2.00E-35 || SODC_CHICK Superoxide dismutase [Cu-Zn] OS=Gallus gallus GN=SOD1 PE=1 SV=3 ||

Trying to construct a discussion for the C.gigas hsp70 situation and figure out the best way to explain what sequences are available, how to differentiate between hsc and hsp70 and what other people have already done...In doing so I've constructed a few alignments from the Kawabe2011 paper which are confusion.
 * May 10, 2011**

Here they are!

Notes: The Kawabe sequences are the qPCR primer sequences AJ318882 is the hsp70 sequence from boutet AB5449340 is the hsp70 sequence used to make the primers from the paper AJ3035315 is the hsc71 sequence from boutet

What's confusing? The hsc primer sequences align better with hsp from boutet, although i would be surprise if they are specific. Their hsp primers appear to fall in the 5'UTR of their published sequence

hsp70 qPCR amplicon sequence analysis
 * May 5, 2011**

__Recap:__ There are discrepancies in the literature regarding the true hsp70 sequence as well as what distinguishes hsp70 from hsc70. How that directly impacts what I"m doing is that I need definitive proof that what I'm measure via qPCR is indeed a single hsp70 sequence given the available data in genbank.

__What did I do?__ - Cloned the PCR products from the two sets of qPCR primers that I designed for hsp70. Each primer pair was designed using different sequences available in genbank. -Sequenced 5 clones for each PCR product as well as directly sequencing the PCR products generated from genomic DNA.

__Sequence analysis summary:__ 1. The product generated from the hsp70 1194 and 1195 primers only amplify the hsp70 corresponding to accession# AB122063. Good news that they are specific, however, this is no considered (at least from my reading) to be the accepted hsp70 sequence for C.gigas.

2. The product generated from the hsp70 RLID 1202 and 1203 primers appear to be specific to the hsp70 sequence corresponding to accession number AJ318882. However, this conclusion is essentially based on a 3bp mismatch between AJ318882 and AJ3035315 (hsc70), which also happens to be the densest accumulation of SNPS that can distinguish the two. Whether this sufficient enough to claim that we are measuring hsp70 only and not detecting any hsc70 transcript is unclear, but this is the most conclusive evidence we can generate to make this argument. Note: I realize that this image is only showing clone#1 but all clones were identical in this region.

3. PCR of genomic DNA using hsp70 primers 1202 and 1203 generated two products. The smaller (expected size) product blasted to AJ318882 while the large band failed to blast to anything in genbank.

Finishing our second attempt at making RAD libraries for M.merc. and quantification of V.tubiashii from May 2, 2011.
 * May 4, 2011**

__RAD library prep Day 2__ Picking up from where we left off last night (blunt repair rxn was put at -20)

1. Purify blunt repair rxn using MiniElute rxn. clean-up kit. - add 300ul buffer PB to sample - apply entire sample to MiniElute column - spin 10,000xg for 1min. discard flow through - add 750ul buffer PE to column - spin 10,000xg for 1min. discard flow through. - spin empty column 10,000xg 1min to remove residual buffer - Transfer column to new tube and apply **42ul** buffer EB to column and incubate 1min at RT. - Spin 10,000xg for 1min. KEEP flow through and discard column.

2. Add A overhang - Make A overhand mastermix (for 1rxn: 5ul NEB2, 1ul dATP, 3ul Klenow) Note: we only have one samples for A overhang components were added directly to the sample. - Incubate at 37 decrees C for 1hr

3. Purify A overhang rxn using MiniElute rxn. clean-up kit. - add 300ul buffer PB to sample - apply entire sample to MiniElute column - spin 10,000xg for 1min. discard flow through - add 750ul buffer PE to column - spin 10,000xg for 1min. discard flow through. - spin empty column 10,000xg 1min to remove residual buffer - Transfer column to new tube and apply **44ul** buffer EB to column and incubate 1min at RT. - Spin 10,000xg for 1min. KEEP flow through and discard column.

4. Ligate P2 Adaptors - Make P2 adaptor master mix (for 1rxn: 5ul NEB2, 1ul P2 Adaptor, 0.5ul rATP, 0.5ul T4 DNA Ligase) Note: again, we only have one sample so components were added directly to sample. - Incubate at RT for 1hr.

5. Purify P2 adaptor rxn using MiniElute rxn. clean-up kit. - add 300ul buffer PB to sample - apply entire sample to MiniElute column - spin 10,000xg for 1min. discard flow through - add 750ul buffer PE to column - spin 10,000xg for 1min. discard flow through. - spin empty column 10,000xg 1min to remove residual buffer - Transfer column to new tube and apply **50ul** buffer EB to column and incubate 1min at RT. - Spin 10,000xg for 1min. KEEP flow through and discard column.

6. Test PCR -Default protocol calls for 1ul of template in the test PCR. Last time we tried this we ended up increasing to 5ul and we still did not acquire enough template. This time I will set up two test PCR rxns. one with 1ul of template and the other with 5ul of template. __1ul Template Test PCR__ 12.5ul Phusion PCR mix 10.5ul Sterile Water 1ul RAD Primer mix 1ul Template

__5ul Template Test PCR__ 12.5ul Phusion PCR mix 6.5ul Sterile Water 1ul RAD Primer mix 5ul Template

Rxn conditions 1. 98 for 30s 2. 98 for 10s 3. 65 for 30s 4.72 for 30s 5. goto 2 15x 6. 72 for 5min 7. hold 4 degrees

It worked! The 5ul rxn was much brighter than the 1ul and the fact that you can easily see the library template is very encouraging. Will move forward with full PCR rxn uping our template to 6ul for an extra boost (hopefully).

__Large RAD PCR rxn.__ 50ul Phusion PCR mix 22ul Sterile Water 4ul RAD primer mix 24ul Template

Rxn conditions were the same as the test PCR above.

On another note... Using Elenes V.tubiashii qPCR protocol to quantify the CFUs from the clam experiment on May 2, 2011

Set up MasterMix for ssoFast Evagreen qPCR mix.
 * Master Mix || 1x || 40 ||
 * 2x Sso Fast EvaGreen || 10 || 400 ||
 * F Primer || 0.5 || 20 ||
 * R Primer || 0.5 || 20 ||
 * Temp. || 2ul ||  ||
 * Water || 7 || 280 ||

May 3, 2011 Starting new RAD library prep for M.merc with a few changes. Notes: last time we tried this we failed to generate enough RAD library template to send for sequencing even though we followed the given protocol exactly. Reasons for this are speculative at best, so we are going to make a few conservative changes in a last ditch effort to to make gobs and gobs for RAD library fragments.

Changes: 1. gDNA from M.merc was precipitated to further clean the samples and remove the NaOH that they were resuspended in according to the DNAzol. Not sure if NaOH alters the downstream reaction chemistries but it's not worth risking. gDNA was resuspended in Qiagen buffer EB.

2. Will perform two Sbf1 digests containing 1ug gDNA for each sample and combine during the rxn. clean up step thus doubling the amount of digested material used from the adaptor ligation on.

3. Pool entire samples from the adaptor ligation for sonication. After pooling, samples will be divided into 120ul aliquots for sonication and then recombined by spinning through a single MiniElute column.

Lets get started!

1. Transferred 1ug of gDNA to a new tube and brought to 40ul. (made two 1ug aliquots for each sample which is a total of 16 rxns) Concentrations were determined by Caroline using PicoGreen assay.
 * Sample || Conc. ng/ul || ul for 1.0ug || ul water for 40ul total ||
 * MA1 || 45.6 || 21.93 || 18.07 ||
 * MA2 || 116.2 || 8.61 || 31.39 ||
 * MA3 || 81.1 || 12.33 || 27.67 ||
 * MA4 || 105.3 || 9.50 || 30.50 ||
 * MAX2 || 140.1 || 7.14 || 32.86 ||
 * MAX3 || 68.7 || 14.56 || 25.44 ||
 * MAX9 || 241.4 || 4.14 || 35.86 ||
 * MAX11 || 228.9 || 4.37 || 35.63 ||

2. Make SbfI mastermix with RNaseA
 * 5/4/11 ||  ||   ||   ||   ||   ||   ||   || + 10% pipetting error ||
 * Number of Individuals: || 16 |||| Concentration ||  ||   ||   ||   ||
 * ||  || Solution || Stock || Final || 1x (ml) || Master Mix ||   ||   ||
 * ||  || NEB4 || 10x || 1x || 5 || 88 ||   ||   ||
 * ||  || H2O || NA || NA || 4 || 70.4 ||   ||   ||
 * ||  || Sbf I - HF || 20 units/ml || 20 units || 1 || 17.6 ||   ||   ||
 * ||  || RNAse A* ||   ||   || 0.1 || 1.76 ||   ||   ||
 * ||  |||||||||||||| 1) Add 10ul to each individual (40ul) sample for a total reaction volume of 50 ul ||
 * ||  |||||||| 2) Incubate at 37 C according to methods ||   ||   ||   ||
 * * Optional See Methods ||  ||   ||   ||   ||   ||   ||   ||
 * ||  |||||||| 2) Incubate at 37 C according to methods ||   ||   ||   ||
 * * Optional See Methods ||  ||   ||   ||   ||   ||   ||   ||
 * * Optional See Methods ||  ||   ||   ||   ||   ||   ||   ||
 * * Optional See Methods ||  ||   ||   ||   ||   ||   ||   ||

2. Heat kill RNase by incubating samples at 65 degrees C for 20min.

3. Clean up reactions using MiniElute rxn. clean up protocol. Note: duplicate samples were pooled to make 8 different 100ul samples which were then processed as follows. - add 300ul buffer PB to sample - apply entire sample to MiniElute column - spin 10,000xg for 1min. discard flow through - add 750ul buffer PE to column - spin 10,000xg for 1min. discard flow through. - spin empty column 10,000xg 1min to remove residual buffer - Transfer column to new tube and apply **47****ul** buffer EB to column and incubate 1min at RT. - Spin 10,000xg for 1min. KEEP flow through and discard column.

4. P1 RAD adaptor Ligation - Add 4ul of P1 RAD Adaptor to each sample (use a different adaptor for each sample) - Add 3ul NEB Buffer 2 to each sample. -Make P1 Ligation mastermix as follows:
 * 5/4/11 ||  ||   ||   ||   ||   |||| + 10% for pipetting error ||
 * Number of Individuals: || 8 ||  ||   ||   ||   ||   ||
 * ||  || Solution || 1x (ul) |||| Master Mix ||   ||   ||
 * ||  || H2O || 3.9 || 34.32 ||   ||   ||   ||
 * ||  || NEB 2 || 2 || 17.6 ||   ||   ||   ||
 * ||  || rATP || 0.6 || 5.28 ||   ||   ||   ||
 * ||  || T4 DNA ligase || 0.5 || 4.4 ||   ||   ||   ||
 * ||  |||||||||| 1) Add 7ul to each individual for a total volume of 60ml ||   ||
 * ||  |||||||| 2) Incubate at Room Temperature for 1hr ||   ||   ||
 * ||  |||||||| 2) Incubate at Room Temperature for 1hr ||   ||   ||

Adaptor Information Note: Obtained adaptor information for the excel sheet that was available in dropbox at the time. Sequences were determined based on plate location, so assuming there is not a more updated information sheet, these should be correct.
 * Sample || Plate well || Barcode sequence || Top oligo sequence || Bottom oligo sequence ||
 * MA1 || B01 || ACTCTT || ACACTCTTTCCCTACACGACGCTCTTCCGATCTACTCTTTGC*A || /5Phos/AAGAGTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT ||
 * MA2 || B02 || ACTGGC || ACACTCTTTCCCTACACGACGCTCTTCCGATCTACTGGCTGC*A || /5Phos/GCCAGTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT ||
 * MA3 || B03 || AGCCAT || ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGCCATTGC*A || /5Phos/ATGGCTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT ||
 * MA4 || B04 || AGCGCA || ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGCGCATGC*A || /5Phos/TGCGCTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT ||
 * MAX2 || B05 || AGGGTC || ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGGGTCTGC*A || /5Phos/GACCCTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT ||
 * MAX3 || B06 || AGGTGT || ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGGTGTTGC*A || /5Phos/ACACCTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT ||
 * MAX9 || B07 || AGTAGG || ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGTAGGTGC*A || /5Phos/CCTACTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT ||
 * MAX11 || B08 || AGTTAA || ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGTTAATGC*A || /5Phos/TTAACTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT ||

5. Heat kill enzyme by incubating at 65 degrees C for 10min.

Note: At this point I had to run to class and Caroline took over proceeded to pool the samples, shear them in the Bioruptor Sonicator, Purified with MiniElute, Gel purify, and begin the Blunt end repair. See Carolines notebook for details!

After the 1hr incubation for the blunt end repair, I moved the rxn to the -20 and will resume the procedure tomorrow.

Clam/Vt experiment
 * May 2, 2011**

Clams were put in seawater on Friday April 29, 2011 3:30pm and were allowed to acclimate to 10 degrees for ~2.5 days. No mortalities occurred during this time.

Clams were separated into four buckets (BX N=10/bucket, MA N=11/bucket). 2L of 10 degree seawater was added to each bucket to just cover the clams.

qPCR test using 18s and hsp70 C.gigas primers and OA larvae cDNA from April 27, 2011
 * April 28, 2011**


 * Master Mix || 1x || 10 ||
 * 2x Sso Fast EvaGreen || 10 || 100 ||
 * F Primer || 0.5 || 5 ||
 * R Primer || 0.5 || 5 ||
 * Temp. || 2ul ||  ||
 * Water || 7 || 70 ||

More RADing and testing and RNA extractions from OA larvae.
 * April 27, 2011**

__C.gigas larval RNA extraction from OA samples.__ Notes: The last time I tried extracting RNA from these samples there was essentially no yield. So what's different this time? I'm going to try extracting another sample from that same sampling date except try using the MRC protocol for low tissue quantities. In addition, I am going to try extracting RNA from the termination samples on April, 18, 2011 since the entire jar was samples instead of a small aliquot. Hopefully this will give me enough RNA to do qPCR, although there was a LOT of algae that came along with the larvae, so I'm not sure if I will actually be extracting gigas RNA or if it is just all going to be algae.

Followed standard TriReagent Protocol Notes: added 8ul of polyacryl carrier to each homogenate, vortexed, and then proceeded with the 5min RT incubation etc. etc.

Results: Notes: Samples 4B1, 5B1, and 6B1 were the termination samples (ie whoe jar) taking on April, 18, 2011. 4B2, 5B2, and 6B2 were taken on April 15, 2011. Yields are slightly better for this second go around however I'm surprised by how little RNA I got from the "termination" samples.
 * Sample ID || User ID || Date || Time || ng/ul || A260 || A280 || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || 340 raw ||
 * 4B1 || Default || 4/27/11 || 11:52 AM || 36.87 || 0.922 || 0.428 || 2.15 || 0.47 || 40 || 230 || 1.976 || 0.029 ||
 * 5B1 || Default || 4/27/11 || 11:53 AM || 27 || 0.675 || 0.296 || 2.28 || 0.25 || 40 || 230 || 2.685 || 0.016 ||
 * 6B1 || Default || 4/27/11 || 11:53 AM || 56.43 || 1.411 || 0.667 || 2.11 || 0.74 || 40 || 230 || 1.914 || 0.049 ||
 * 4B2 || Default || 4/27/11 || 11:54 AM || 11.47 || 0.287 || 0.157 || 1.82 || 0.2 || 40 || 230 || 1.405 || 0.076 ||
 * 5B2 || Default || 4/27/11 || 11:54 AM || 5.72 || 0.143 || 0.076 || 1.89 || 0.23 || 40 || 230 || 0.636 || 0.024 ||
 * 6B2 || Default || 4/27/11 || 11:55 AM || 26.08 || 0.652 || 0.309 || 2.11 || 0.16 || 40 || 230 || 4.06 || 0.057 ||

I'm curious about how much of the RNA that I isolated is algae vs gigas...in other words is it useful? I took samples 4B1 5B1 and 6B1 and proceeded with ant RT reaction for subsequent qPCR. I'll just try amplifying 18s and hsp70 for fun to see if I get anything...

__Hard Clam RAD Libraries__ Caroline set up the test PCR last night.

-Ran PCR on 1% agarose gel to check amplifiation Results: Cant see anything After talking with members of the Seeb lab, the DNA quantities that we are dealing with are so low that it might be impossible to see them on a standard agarose gel. They suggest running our PCR samples on one of their EGels...they are much smaller/thinner then what we can pour so any DNA present whould stand out more easily.

-Ran a 1% EGel Results: See Carolines Notebook.

Troubleshooting: One possible cause for our PCR failure is that the concentration of our fragment libraries is too low. I still have the set of individual barcode ligations frozen away in the -20. We are going to try fragmenting TWICE as much DNA as recommended by the protocol to see if that yields better results. This means going back to the pooling step followed by sonication etc etc.

RAD protocol continued.
 * April 26, 2011**

-__Gel Purification__ 1. Ran sample from April 25, 2011 on a 1% agarose gel with both an NEB 1KB ladder (N3231) and an EGel 1kb ladder (was not sure which one would give me the best resolution at the 300-700bp range for subsequent size isolation.) 2. Cut our gel size fraction between 350-650bp. Band sizes for NEB 1KB ladder N3231

3. Use minielute gel purifiation protocolt to purify fragments. -Gel weight = 400mg. added 1,200ul of Buffer QG and incubated at 50 degrees C for 10min to dissolve gel frag. - Added 400ul isopropanol and spun through minielute column (800ul at at time until all was spun through) - Added 500ul buffer QG to column and spun 1min 13K rpm - Added 750ul buffer PE and spun 1min at 13K rpm - Discard flow-though and spun again for 1min at 13K rpm - Added 20ul Buffer EB directly to column. Incubated 1min at RT and spun 1min at 13K rpm.

Beginning RAD protocol for hard clam QPX samples.
 * April 25, 2011**

Sbf-1 Digest: Transfered 1ug DNA to a new tube and brought volume to 40ul with sterile water Prepared digest master mix using the excel worksheet. Included RNase A digest. - Added 10ul of master mix to each sample and incubated at 37 degrees C for 1hr - Heat Kill enzyme by incubating at 65 degrees C for 20min
 * Sample ID || avg. conc. || ul for 1ug ||
 * MAX 1 || 177.06 || 5.65 ||
 * MAX 2 || 379.93 || 2.63 ||
 * MAX 3 || 190.33 || 5.25 ||
 * MAX 4 || 86.29 || 11.59 ||
 * MA 1 || 116.35 || 8.60 ||
 * MA 2 || 271.01 || 3.69 ||
 * MA 3 || 195.89 || 5.10 ||
 * MA 4 || 245.76 || 4.07 ||
 * FL3_1 || 353.73 || 2.83 ||
 * FL3_3 || 435.43 || 2.30 ||
 * FL3_4 || 152.68 || 6.55 ||
 * FL3_5 || 229.33 || 4.36 ||
 * 4/25/11 ||  ||   ||   ||   ||   |||| All reactions + 10% for pipetting error ||
 * Number of Individuals: || 12 ||  ||   ||   ||   ||   ||
 * ||  || Solution || 1x (ml) |||| Master Mix ||   ||   ||
 * ||  || NEB4 || 5 || 66 ||   ||   ||   ||
 * ||  || H2O || 4 || 52.8 ||   ||   ||   ||
 * ||  || Sbf I - HF || 1 || 13.2 ||   ||   ||   ||
 * ||  || RNAse A* || 0.1 || 1.32 ||   ||   ||   ||
 * ||  |||||| 1) Add 10ml to each individual ||   ||   ||   ||
 * ||  |||||||| 2) Incubate at 37 C according to methods ||   ||   ||
 * * Optional See Methods ||  ||   ||   ||   ||   ||   ||
 * * Optional See Methods ||  ||   ||   ||   ||   ||   ||
 * * Optional See Methods ||  ||   ||   ||   ||   ||   ||
 * * Optional See Methods ||  ||   ||   ||   ||   ||   ||

__- MiniElute Clean-up__ 1. Add 300ul Buffer PB to sample 2. Spin sample through MiniElute column for 1min at 13K rpm 3. Discard flow-through and add 750ul wash buffer PE to column. 4. Spin 1min at 13K rpm 5. Discard flow-through and spin again 1min 13K rpm to remove residual wash buffer. 6. Elute in 47ul buffer EB by applying directly to column. Incubate RT 1min followed by 1min sping at 13K rpm.

__-RAD adapter Ligation__ 1. Add 4ul P1 RAD Adapter and 3ul NEB Buffer 2. NOTE: Used RAD adapters A1-A12 on adapter plate for samples MA1-4, MAX 1-4, and FL3-1 - 3-5 in that order. 2. Add 7ul P1 Ligation Mastermix to each sample. 3. Incubate at RT for 1hr. 4. Heat kill enzyme by incubating at 65 degrees C for 10min.

__-Pool Samples__ -Pooled equal amounts (10ul) of each tagged sample for a total of 120ul. -Stored remaining sample at -20 in "Hard Clam gDNA and RAD samples"

__-Shear Pooled Samples__ -Turn on water bath and sonicator at least 30min prior to use to give the water bath a chance to cool to 4 degrees C - NOTE: Before turning on water batch make sure valve in rear of chiller is open to allow water to flow between sonicator bath and chiller bath. - To shear: Run two cycles of 3x 30s sonication, 60s break followed by 1 cycle of 4x 30s sonication, 60s break. Remove samples and spin down between each cycle. -Purify in minielute column.

NOTE: Stored samples o/n at -20

Requantifying gDNA isolated on April 17, 2011 using the nanodrop from the young lab. Apparently our nanodrop is currently not calibrated. Quantification was done with the helpful hand of Caroline Storer aka C-dogg.
 * April 21, 2011**

Only 4 samples were quantified because we are going to run a single multiplex of 12 samples to test our RAD protocol/technique. Each sample was measured 3 times.
 * Sample ID || Date || Time || ng/ul || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || A340 ||
 * MAX 1 || 4/21/2011 || 12:46 PM || 177.33 || 1.91 || 1.55 || 50 || 230 || 2.289 || 0.306 ||
 * MAX 1 || 4/21/2011 || 12:47 PM || 176.45 || 1.91 || 1.59 || 50 || 230 || 2.225 || 0.279 ||
 * MAX 1 || 4/21/2011 || 12:47 PM || 177.41 || 1.92 || 1.57 || 50 || 230 || 2.263 || 0.291 ||
 * MAX 2 || 4/21/2011 || 12:48 PM || 381.26 || 1.86 || 1.31 || 50 || 230 || 5.805 || 0.894 ||
 * MAX 2 || 4/21/2011 || 12:48 PM || 381.08 || 1.85 || 1.33 || 50 || 230 || 5.741 || 0.83 ||
 * MAX 2 || 4/21/2011 || 12:49 PM || 377.46 || 1.86 || 1.35 || 50 || 230 || 5.582 || 0.738 ||
 * MAX 3 || 4/21/2011 || 12:49 PM || 190.69 || 1.94 || 1.66 || 50 || 230 || 2.298 || 0.267 ||
 * MAX 3 || 4/21/2011 || 12:50 PM || 190.37 || 1.95 || 1.66 || 50 || 230 || 2.293 || 0.272 ||
 * MAX 3 || 4/21/2011 || 12:50 PM || 189.94 || 1.94 || 1.69 || 50 || 230 || 2.246 || 0.261 ||
 * MAX 4 || 4/21/2011 || 12:51 PM || 84.07 || 2.02 || 2.06 || 50 || 230 || 0.818 || 0.115 ||
 * MAX 4 || 4/21/2011 || 12:52 PM || 86.29 || 1.99 || 2.15 || 50 || 230 || 0.804 || 0.09 ||
 * MAX 4 || 4/21/2011 || 12:52 PM || 88.5 || 1.96 || 2.02 || 50 || 230 || 0.876 || 0.12 ||
 * MA 1 || 4/21/2011 || 12:53 PM || 116.07 || 1.96 || 1.85 || 50 || 230 || 1.253 || 0.132 ||
 * MA 1 || 4/21/2011 || 12:54 PM || 117.24 || 1.96 || 1.9 || 50 || 230 || 1.232 || 0.126 ||
 * MA 1 || 4/21/2011 || 12:54 PM || 115.73 || 1.96 || 1.96 || 50 || 230 || 1.183 || 0.117 ||
 * MA 2 || 4/21/2011 || 12:55 PM || 271.78 || 1.91 || 1.58 || 50 || 230 || 3.44 || 0.366 ||
 * MA 2 || 4/21/2011 || 12:55 PM || 270.29 || 1.92 || 1.65 || 50 || 230 || 3.285 || 0.316 ||
 * MA 2 || 4/21/2011 || 12:56 PM || 270.97 || 1.91 || 1.65 || 50 || 230 || 3.275 || 0.339 ||
 * MA 3 || 4/21/2011 || 12:57 PM || 195.02 || 1.96 || 1.82 || 50 || 230 || 2.149 || 0.179 ||
 * MA 3 || 4/21/2011 || 12:57 PM || 196.34 || 1.94 || 1.81 || 50 || 230 || 2.17 || 0.218 ||
 * MA 3 || 4/21/2011 || 12:58 PM || 195.74 || 1.95 || 1.78 || 50 || 230 || 2.198 || 0.197 ||
 * MA 3 || 4/21/2011 || 12:58 PM || 196.46 || 1.94 || 1.81 || 50 || 230 || 2.169 || 0.214 ||
 * MA 4 || 4/21/2011 || 12:59 PM || 244.85 || 1.91 || 1.56 || 50 || 230 || 3.147 || 0.271 ||
 * MA 4 || 4/21/2011 || 1:00 PM || 245.96 || 1.91 || 1.54 || 50 || 230 || 3.198 || 0.293 ||
 * MA 4 || 4/21/2011 || 1:00 PM || 246.46 || 1.9 || 1.52 || 50 || 230 || 3.242 || 0.312 ||
 * FL3_1 || 4/21/2011 || 1:01 PM || 352.73 || 1.96 || 0.95 || 50 || 230 || 7.432 || 0.328 ||
 * FL3_1 || 4/21/2011 || 1:02 PM || 353.31 || 1.96 || 0.94 || 50 || 230 || 7.492 || 0.345 ||
 * FL3_1 || 4/21/2011 || 1:03 PM || 355.15 || 1.96 || 0.94 || 50 || 230 || 7.539 || 0.336 ||
 * FL3_3 || 4/21/2011 || 1:03 PM || 438.34 || 1.93 || 1.16 || 50 || 230 || 7.533 || 0.509 ||
 * FL3_3 || 4/21/2011 || 1:04 PM || 429.53 || 1.93 || 1.17 || 50 || 230 || 7.318 || -0.703 ||
 * FL3_3 || 4/21/2011 || 1:05 PM || 430.49 || 1.94 || 1.16 || 50 || 230 || 7.396 || 0.472 ||
 * FL3_3 || 4/21/2011 || 1:06 PM || 443.37 || 1.92 || 1.11 || 50 || 230 || 7.967 || 0.592 ||
 * FL3_4 || 4/21/2011 || 1:07 PM || 152.88 || 1.86 || 0.87 || 50 || 230 || 3.497 || 0.445 ||
 * FL3_4 || 4/21/2011 || 1:08 PM || 156.95 || 1.87 || 0.88 || 50 || 230 || 3.562 || 0.363 ||
 * FL3_4 || 4/21/2011 || 1:08 PM || 148.22 || 1.9 || 0.98 || 50 || 230 || 3.015 || 0.261 ||
 * FL3_5 || 4/21/2011 || 1:09 PM || 228.81 || 2.02 || 0.42 || 50 || 230 || 10.813 || 0.203 ||
 * FL3_5 || 4/21/2011 || 1:10 PM || 228.31 || 2.03 || 0.42 || 50 || 230 || 10.81 || 0.187 ||
 * FL3_5 || 4/21/2011 || 1:10 PM || 230.88 || 2.02 || 0.43 || 50 || 230 || 10.863 || 0.224 ||

Transfered Cgigas larvae from the OA sampling on April 18, 2011 from RNAlater to 800ul TriReagent as recommened by the MRC protocol for small tissue samples

April 19, 2011 Quantification of gDNA from April 17, 2011 extractions using nanodrop.


 * Sample ID || User ID || Date || Time || ng/ul || A260 || A280 || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || 340 raw ||
 * MA 1 || Default || 4/19/11 || 9:33 AM || 99.37 || 1.987 || 1.072 || 1.85 || 2.28 || 50 || 230 || 0.871 || 0.082 ||
 * MA 2 || Default || 4/19/11 || 9:34 AM || 234.86 || 4.697 || 2.585 || 1.82 || 1.73 || 50 || 230 || 2.722 || 0.297 ||
 * MA 3 || Default || 4/19/11 || 9:34 AM || 171.63 || 3.433 || 1.85 || 1.85 || 1.92 || 50 || 230 || 1.783 || 0.168 ||
 * MA 4 || Default || 4/19/11 || 9:35 AM || 211.83 || 4.237 || 2.311 || 1.83 || 1.59 || 50 || 230 || 2.659 || 0.287 ||
 * MA 5 || Default || 4/19/11 || 9:35 AM || 153.87 || 3.077 || 1.66 || 1.85 || 1.8 || 50 || 230 || 1.711 || 0.186 ||
 * MA 6 || Default || 4/19/11 || 9:36 AM || 77.49 || 1.55 || 0.838 || 1.85 || 2.74 || 50 || 230 || 0.565 || 0.067 ||
 * MA 7 || Default || 4/19/11 || 9:36 AM || 135.88 || 2.718 || 1.472 || 1.85 || 1.98 || 50 || 230 || 1.376 || 0.138 ||
 * MA 8 || Default || 4/19/11 || 9:37 AM || 90.8 || 1.816 || 0.958 || 1.89 || 2.08 || 50 || 230 || 0.873 || 0.136 ||
 * MA 9 || Default || 4/19/11 || 9:37 AM || 236.73 || 4.735 || 2.674 || 1.77 || 1.11 || 50 || 230 || 4.247 || 1.117 ||
 * MA 10 || Default || 4/19/11 || 9:39 AM || 83.9 || 1.678 || 0.907 || 1.85 || 1.88 || 50 || 230 || 0.89 || 0.115 ||
 * MA 11 || Default || 4/19/11 || 9:39 AM || 100.34 || 2.007 || 1.086 || 1.85 || 1.71 || 50 || 230 || 1.175 || 0.166 ||
 * MA 12 || Default || 4/19/11 || 9:40 AM || 92.97 || 1.859 || 0.979 || 1.9 || 2.23 || 50 || 230 || 0.833 || 0.102 ||
 * MA 13 || Default || 4/19/11 || 9:40 AM || 272.73 || 5.455 || 2.957 || 1.84 || 1.61 || 50 || 230 || 3.393 || 0.338 ||
 * MA 14 || Default || 4/19/11 || 9:41 AM || 226.01 || 4.52 || 2.434 || 1.86 || 1.77 || 50 || 230 || 2.56 || 0.267 ||
 * MA 15 || Default || 4/19/11 || 9:41 AM || 112.11 || 2.242 || 1.178 || 1.9 || 2.61 || 50 || 230 || 0.859 || 0.041 ||
 * MAX 1 || Default || 4/19/11 || 9:43 AM || 167.04 || 3.341 || 1.842 || 1.81 || 1.48 || 50 || 230 || 2.254 || 0.309 ||
 * MAX 2 || Default || 4/19/11 || 9:43 AM || 324.42 || 6.488 || 3.612 || 1.8 || 1.38 || 50 || 230 || 4.718 || 0.668 ||
 * MAX 3 || Default || 4/19/11 || 9:44 AM || 172.88 || 3.458 || 1.879 || 1.84 || 1.73 || 50 || 230 || 1.999 || 0.248 ||
 * MAX 4 || Default || 4/19/11 || 9:44 AM || 79.51 || 1.59 || 0.835 || 1.9 || 2.49 || 50 || 230 || 0.64 || 0.085 ||
 * MAX 5 || Default || 4/19/11 || 9:45 AM || 182.09 || 3.642 || 1.993 || 1.83 || 1.5 || 50 || 230 || 2.436 || 0.294 ||
 * MAX 6 || Default || 4/19/11 || 9:45 AM || 127.69 || 2.554 || 1.382 || 1.85 || 1.97 || 50 || 230 || 1.295 || 0.146 ||
 * MAX 7 || Default || 4/19/11 || 9:46 AM || 103.07 || 2.061 || 1.111 || 1.86 || 2.42 || 50 || 230 || 0.852 || 0.06 ||
 * MAX 8 || Default || 4/19/11 || 9:50 AM || 267.78 || 5.356 || 2.976 || 1.8 || 1.38 || 50 || 230 || 3.872 || 0.591 ||
 * MAX 9 || Default || 4/19/11 || 9:47 AM || 335.44 || 6.709 || 3.721 || 1.8 || 1.39 || 50 || 230 || 4.834 || 0.61 ||
 * MAX 10 || Default || 4/19/11 || 9:48 AM || 176.74 || 3.535 || 1.913 || 1.85 || 1.77 || 50 || 230 || 1.996 || 0.174 ||
 * MAX 11 || Default || 4/19/11 || 9:48 AM || 359.04 || 7.181 || 3.999 || 1.8 || 1.41 || 50 || 230 || 5.089 || 0.669 ||
 * MAX 12 || Default || 4/19/11 || 9:48 AM || 241.85 || 4.837 || 2.698 || 1.79 || 1.22 || 50 || 230 || 3.975 || 0.837 ||
 * MAX 13 || Default || 4/19/11 || 9:49 AM || 189.57 || 3.791 || 2.088 || 1.82 || 1.38 || 50 || 230 || 2.755 || 0.271 ||
 * MAX 14 || Default || 4/19/11 || 9:49 AM || 121.67 || 2.433 || 1.307 || 1.86 || 1.76 || 50 || 230 || 1.385 || 0.179 ||
 * MAX 15 || Default || 4/19/11 || 9:50 AM || 355.28 || 7.106 || 3.994 || 1.78 || 1.26 || 50 || 230 || 5.65 || 0.892 ||

Termination of C.gigas larval OA experiment from April 11, 2011.
 * April 18, 2011**

Sampling notes: Adjusted live/dead count sampling volumes to 3x 33ul aliquots to account for decreased larval densities.

All remaining larve were isolated using a 40micron cell strainer and preserved in RNAlater for subsequent RNA isolation.

Today I finished the DNAzol extractions that I started yesterday...almost. They are currently re-suspending at 4 degrees.
 * April 17, 2011**

Spun PK digests 10,000xg for 10min. Transfered supt. to new tube. Note: tissue was not "dissolved" and still looked like gill tissue, only it was much clearer than what I started with yesterday. Added 250ul of 100% EtOH and incubated at RT for 3min. Spun down gDNA at 5,000xg for 5min Discard supt. Wash 2x 1ml 75% EtOH with 2min spin at 1,000xg between washes Resuspended in 200ul 8mM NaOH pH8. pH was adjusted by adding 100ul of .1M HEPES/1ml 8mM NaOH.

DNAzol extractions of QPX clam samples for RAD sequencing. Extrating MA (Mashpee) 1-15 and MAX (Barnstable) 1-15 rec'd Nov. 2009 from Rutgers.
 * April 16, 2011**

Started gDNA extractions. Added 500ul DNAzol to ~30mg pieces of Mm gill tissue + 2.7ul 18.3mg/ml Proteinase K (100ug/ml final conc.) Inucbated o/n at RT with inversion.

More sampling at NOAA and testing New genomics sampling technique.
 * April 15, 2011 **

New sampling technique: I found these "cell strainer" baskets in the Friedman lab that brent said I would try out. Basically they are 40micron mesh basket screens that are made to fit in a 6 well tissue culture plate. The idea for sampling is to filter the larvae into the basket and then submerge the strainer in a 6 well plate containing RNA later. Once back to the lab, the strainer can be removed from well containing RNA later and transferred to a well containing 1ml of TriReagent. A pipet can then be used to apply the TriReagent to the strainer effectively lysing the cells and washing them off of the straining. TriReagent homogenate can then me transferred to a centrifuge tube and the rest of the TriReagent protocol can be carrie out as normal.

Sampling notes: Took two replicates of 3 x 50ul aliquots of larvae from 50ml falcon tube for live/dead counts. Took 5ml from 50ml falcon tube for genomics sample.

Extracted 4B3, 5B3, and 6B3 to test new sampling method.

Results: Basically nothing. 4B3 shows some promise but something still isnt working right. First I want to see the final counts from emma so I can estimate the number of larvae I extracted.
 * Sample ID || User ID || Date || Time || ng/ul || A260 || A280 || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || 340 raw ||
 * 4B3 || Default || 4/15/11 || 2:56 PM || 2.5 || 0.062 || 0.019 || 3.35 || 1.55 || 40 || 230 || 0.04 || -0.007 ||
 * 5B3 || Default || 4/15/11 || 2:57 PM || 18.01 || 0.45 || 0.29 || 1.55 || 2.52 || 40 || 230 || 0.179 || 0.017 ||
 * 6B3 || Default || 4/15/11 || 2:58 PM || 4.29 || 0.107 || 0.028 || 3.84 || 0.1 || 40 || 230 || 1.036 || 0.001 ||

RNA extractions to test samples from April 12, 2011
 * April 14, 2011 **

Samples were thawed and spun at 4 degrees for 2min to pellet the larvae. Followed standard TriReagent protocol for RNA extraction

Results: Nothing. Nanodrop read ~5ng/ul which is within range of normal bacground readings. In addition, absorbance ratios are way off. Ideas: Could have lost a lot of larvae that stuck to the filter when we tried to remove the saltwater. Freezing samples in saltwater could have negative effects on RNA quality. Just didnt have enough larvae.
 * Sample ID || User ID || Date || Time || ng/ul || A260 || A280 || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || 340 raw ||
 * 4B1 || Default || 4/14/11 || 10:08 AM || 6.06 || 0.152 || 0.104 || 1.46 || 0.36 || 40 || 230 || 0.422 || -0.006 ||
 * 5B1 || Default || 4/14/11 || 10:09 AM || 5.5 || 0.137 || 0.102 || 1.34 || 0.38 || 40 || 230 || 0.358 || -0.015 ||
 * 6B1 || Default || 4/14/11 || 10:09 AM || 6.56 || 0.164 || 0.11 || 1.5 || 0.21 || 40 || 230 || 0.787 || -0.002 ||

Today we (Sam, Emma, and myself) spent the morning at NOAA taking samples of C.gigas larvae for an OA experiment.
 * April 12, 2011 **

Experimental Notes: 3 Different pCO2 treatments: 380, 540, and 1000 3 replicate jars/ treatment Eggs were fertilized on April 11, 2011 around 12:00 noon and collected at 2:30pm. Larvae went into the NOAA system around 5:30 pm and sat static until the following morning. Sampling began around 9am on April 12, 2011. Once complete, larval concentrations were adjusted based on counts and jars were hooked up to the system for the first time around 2:00pm.

Sampling Notes: Larval jars were filtered and condensed into 50ml falcon tubes as described by the diagram from April 7, 2011. Two replicates of three 30ul aliquots were taken from the 50ml falcon tube for live/dead counts. 750ul of larvae were sampled for genomics by filtering as much seawater away as possible and snap freezing in LN2. After filtering, the final volume of seawater and larvae was ~200ul.

Today I spent the day over at NOAA to see how they were sampling their geoduck larvae. Below is a diagram I made as a visual aid to the whole process.
 * April 7, 2011**

Notes: The entire process was carried out on the first jar as a test to see what aliquot volume would yield ~50 larvae. At a starting concentration of 1 larvae/ml, ~30ul was taken/aliquot.

A total of two replicates was taken from each jar for counting.

Use Qiagen DNeasy miniprep kits to isolate plasmid from cultures started on April 1, 2011. Resuspended plasmid DNA in 50ul sterile water.
 * April 4, 2011**

Plasmid DNA looks good. Waiting for sequencing plate to fill so we can send them off for sequencing.
 * Sample ID || User ID || Date || Time || ng/ul || A260 || A280 || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || 340 raw ||
 * hps70OldPrimers Clone 1 || Default || 4/4/11 || 1:00 PM || 350.42 || 7.008 || 3.725 || 1.88 || 2.27 || 50 || 230 || 3.089 || 0.023 ||
 * hsp70OldPrimers Clone 2 || Default || 4/4/11 || 1:01 PM || 285.92 || 5.718 || 3.038 || 1.88 || 2.28 || 50 || 230 || 2.507 || 0.016 ||
 * hsp70OldPrimers Clone 3 || Default || 4/4/11 || 1:01 PM || 332.78 || 6.656 || 3.537 || 1.88 || 2.24 || 50 || 230 || 2.966 || -0.006 ||
 * hsp70OldPrimers Clone 4 || Default || 4/4/11 || 1:02 PM || 361.23 || 7.225 || 3.82 || 1.89 || 2.31 || 50 || 230 || 3.124 || -0.015 ||
 * hsp70OldPrimers Clone 5 || Default || 4/4/11 || 1:02 PM || 214.82 || 4.296 || 2.266 || 1.9 || 2.26 || 50 || 230 || 1.902 || 0.028 ||
 * hsp70NewPrimers Clone 1 || Default || 4/4/11 || 1:03 PM || 325.87 || 6.517 || 3.459 || 1.88 || 2.3 || 50 || 230 || 2.838 || 0.019 ||
 * hsp70NewPrimers Clone 2 || Default || 4/4/11 || 1:03 PM || 292.58 || 5.852 || 3.085 || 1.9 || 2.34 || 50 || 230 || 2.505 || -0.025 ||
 * hsp70NewPrimers Clone 3 || Default || 4/4/11 || 1:04 PM || 333.7 || 6.674 || 3.535 || 1.89 || 2.18 || 50 || 230 || 3.068 || 0.032 ||
 * hsp70NewPrimers Clone 4 || Default || 4/4/11 || 1:04 PM || 250.44 || 5.009 || 2.7 || 1.86 || 1.81 || 50 || 230 || 2.773 || 0.359 ||
 * hsp70NewPrimers Clone 5 || Default || 4/4/11 || 1:05 PM || 307.85 || 6.157 || 3.261 || 1.89 || 2.16 || 50 || 230 || 2.844 || 0.095 ||

Made glycerol stocks from 5ml o/n cultures started on April 1, 2011 and transferred the remaining cultures to 4 degrees until I have a chance to do minipreps.
 * April 2, 2011**

Cloning hsp70 amplicons for sequencing. O/N plates grew extremely well and I have tons of colonies to choose from. Picked colonies into 20ul sterile water. Used 2ul of colony/water mixture as PCR template
 * April 1, 2011 **

PCR: MasterMix: 12.5ul M13 F Primer (10uM): 0.5ul M13 R Primer (10uM): 0.5ul Water: 9.5ul Template: 2ul

Ran same cycling conditions as March 31, 2011 except only ran 30 cycles instead of 40.

Gel Loading and Naming: 1-N = "Old" hsp70 primers - clone # 2-N = "New hsp70 primers - clone #

Lane 1: Ladder Lane 2: Positive control Lane 3: 1-1 Lane 4: 1-2 Lane 5: 1-3 Lane 6 1-4 Lane 7: 1-5 Lane 8: Negative control Lane 9: Positive control Lane 10: 2-1 Lane 11: 2-2 Lane 12: 2-3 Lane 13: 2-4 Lane 14: 2-5 Lane 15: Negative control

Results: All clones are positive for the appropriate insert of the appropriate size. Started 5ml o/n cultures to make glycerol stocks and extract plasmid DNA for sequencing.

Started 5ml o/n cultures in LB + Kan for subsequent plasmid isolation.

hsp70 PCR with "old" and "new" primers for subsequent cloning and sequencing to verify qPCR amplicon.
 * March 31, 2011 **

PCR mix 2x MasterMix: 2ul F Primer (10uM): 0.5ul R Primer (10uM): 0.5ul Water: 9.5ul cDNA template: 2ul

Cycling conditions: 1. 95 for 10min 2. 95 for 15sec 3. 55 for 30sec 4. 72 for 30sec 5. Goto 2 39x 6. 72 for 10min 7. 4 forever (these conditions mimic those used for qPCR minus the 72deg for 10min final extension.) Lane 1: Ladder Lane 2: "Old" hsp70 primer pair Lane 3: - control for "old" primer pair Lane 4: "New" hsp70 primer pair Lane 5: - control for "new" primer pair

Results: PCR looks good. Cut out the bands and extracted using millipore spin columns (5000xg for 10min) for cloning.

Cloning: Followed standard cloning protocol for Invitrogen Topo TA cloning with Top10 competent cells. Used 4ul of purified PCR product in ligation reaction.


 * March 18, 2011**

Testing "new" hsp70 qPCR primers for a) functionality and b) ability to detect genomic carryover. The "new" hsp70 primers refer to primers 1202 & 1203 in the primer database designed on March, 16, 2011
 * hsp70 qPCR using primers to the Boutet et al. sequence for hsp70**

Ran a qPCR containing RNA samples to test for detectability of DNA carryover and a cDNA sample as a positive control. Everything looks great! Unable to detect DNA carry over and signal for cDNA is strong with a good looking melting curve. For Raw data click here (link coming soon....)

Since the test went well, I'm going to go ahead and run the complete set of CuVt cDNA. Used same mastermix volumes as January 21, 2011 except used primers 1202 & 1203 from above.

Note: removed one outlier from CuVt...expression was "48." There was also an outlier in the protein data. Need to check if they are the same sample...

Extracted RNA from more gill tissues from the hard clam/QPX project using TriReagent. Extracted samples and total RNA concentrations can be found in the table below. Samples correspond to the Scudders lane samples received from MBL on 9/2/10 MA - Mashpee BX - Barnstable Two samples were extracted from each individual plot of a total of 8 samples/strain. RNA was resuspended in 50ul water.
 * March 17, 2011 **


 * Sample ID || User ID || Date || Time || ng/ul || A260 || A280 || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || 340 raw ||
 * MA 1-1 || Default || 3/17/11 || 12:11 PM || 404.84 || 10.121 || 5.651 || 1.79 || 2.24 || 40 || 230 || 4.509 || 0.084 ||
 * MA 1-2 || Default || 3/17/11 || 12:12 PM || 157.85 || 3.946 || 2.267 || 1.74 || 2.3 || 40 || 230 || 1.714 || 0.059 ||
 * MA 2-1 || Default || 3/17/11 || 12:13 PM || 1073.06 || 26.827 || 14.176 || 1.89 || 2.33 || 40 || 230 || 11.498 || 0.125 ||
 * MA 2-2 || Default || 3/17/11 || 12:13 PM || 919.03 || 22.976 || 11.984 || 1.92 || 2.3 || 40 || 230 || 9.99 || 0.244 ||
 * MA 3-1 || Default || 3/17/11 || 12:14 PM || 1247.29 || 31.182 || 16.226 || 1.92 || 2.16 || 40 || 230 || 14.405 || 0.236 ||
 * MA 3-2 || Default || 3/17/11 || 12:14 PM || 364.35 || 9.109 || 4.993 || 1.82 || 2.14 || 40 || 230 || 4.263 || 0.093 ||
 * MA 4-1 || Default || 3/17/11 || 12:15 PM || 430.05 || 10.751 || 5.985 || 1.8 || 2.24 || 40 || 230 || 4.8 || 0.103 ||
 * MA 4-2 || Default || 3/17/11 || 12:15 PM || 403.81 || 10.095 || 5.653 || 1.79 || 2.34 || 40 || 230 || 4.313 || 0.077 ||
 * BX 1-1 || Default || 3/17/11 || 12:16 PM || 676.93 || 16.923 || 8.92 || 1.9 || 2.14 || 40 || 230 || 7.896 || 0.323 ||
 * BX 1-2 || Default || 3/17/11 || 12:16 PM || 565.74 || 14.144 || 7.626 || 1.85 || 2.18 || 40 || 230 || 6.492 || 0.224 ||
 * BX 2-1 || Default || 3/17/11 || 12:17 PM || 278.76 || 6.969 || 3.924 || 1.78 || 2.29 || 40 || 230 || 3.038 || 0.044 ||
 * BX 2-2 || Default || 3/17/11 || 12:18 PM || 612.32 || 15.308 || 8.202 || 1.87 || 2.32 || 40 || 230 || 6.612 || 0.157 ||
 * BX 3-1 || Default || 3/17/11 || 12:18 PM || 126.41 || 3.16 || 1.858 || 1.7 || 0.51 || 40 || 230 || 6.189 || 1.179 ||
 * BX 3-2 || Default || 3/17/11 || 12:19 PM || 459.08 || 11.477 || 6.555 || 1.75 || 2.23 || 40 || 230 || 5.138 || 0.124 ||
 * BX 4-1 || Default || 3/17/11 || 12:19 PM || 298.19 || 7.455 || 4.17 || 1.79 || 2.26 || 40 || 230 || 3.292 || 0.061 ||
 * BX 4-2 || Default || 3/17/11 || 12:20 PM || 1028.29 || 25.707 || 13.519 || 1.9 || 2.29 || 40 || 230 || 11.232 || 0.227 ||

Found out from Sam that the place we use to make our SOLiD libraries uses the same Poly-A isolation kit. New plan is to pool equal amounts of total RNA from all 8 samples and send total RNA pools. Volumes of RNA used to pool can be found in the following table. Pooled samples were DNased treated (30ug total RNA = 3x scaled DNase rxn).


 * Sample ID || ng/ul || ul RNA ||
 * MA 1-1 || 404.84 || 9.26 ||
 * MA 1-2 || 157.85 || 23.76 ||
 * MA 2-1 || 1073.06 || 3.49 ||
 * MA 2-2 || 919.03 || 4.08 ||
 * MA 3-1 || 1247.29 || 3.01 ||
 * MA 3-2 || 364.35 || 10.29 ||
 * MA 4-1 || 430.05 || 8.72 ||
 * MA 4-2 || 403.81 || 9.29 ||
 * BX 1-1 || 676.93 || 5.54 ||
 * BX 1-2 || 565.74 || 6.63 ||
 * BX 2-1 || 278.76 || 13.45 ||
 * BX 2-2 || 612.32 || 6.12 ||
 * BX 3-1 || 126.41 || 29.67 ||
 * BX 3-2 || 459.08 || 8.17 ||
 * BX 4-1 || 298.19 || 12.58 ||
 * BX 4-2 || 1028.29 || 3.65 ||

Final nanodrop specs for pooled DNased sampels are as follows:

Samples are in ~150ul which means there is ~21ug total RNA for each sample.
 * Sample ID || User ID || Date || Time || ng/ul || A260 || A280 || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || 340 raw ||
 * MA pool || Default || 3/17/11 || 2:19 PM || 140.49 || 3.512 || 1.786 || 1.97 || 1.89 || 40 || 230 || 1.855 || 0.02 ||
 * BX pool || Default || 3/17/11 || 2:20 PM || 138.63 || 3.466 || 1.8 || 1.93 || 1.36 || 40 || 230 || 2.541 || 0.143 ||

Pooling Lisa's abalone samples for SOLiD libraries Trying to expedite getting the SOLiD libraries made, I went ahead and pooled lisa/Sam's abalone samples because Sam has been home sick and will be unable to deal with this until next week.

After much deliberation with Sam, I ended up pooling the following quantities of totalRNA samples that have NOT been DNased. Pooling of these samples exhausted the majority of the remaining totalRNA stock, however I found ~30-50ul of DNased RNA for each sample that can be used to synthesize cDNA or whatever.


 * Accession # || who am I? || Sample Type || RNA Extracted || [RNA] (ug/ul) || uL RNA needed for 5.0ug ||
 * 08:4-03 || SC || Termination || 4/2/09 || 0.40838 || 12.98 ||
 * 08:4-04 || SC || Termination || 4/2/09 || 0.66677 || 7.95 ||
 * 08:4-05 || SC || Termination || 4/2/09 || 0.90708 || 5.84 ||
 * 08:4-06 || SC || Termination || 6/3/09 || 0.24997 || 21.2 ||
 * 08:4-13 || SC || Termination || 6/4/09 || 0.28052 || 18.89 ||
 * 08:4-10 || SC || Termination || 6/4/09 ||  || 18.47 ||
 * ||  ||   ||   ||   || 91.27 ||
 * 08:3-11 || SE || Termination || 3/27/09 || 0.34282 || 15.46 ||
 * 08:3-12 || SE || Termination || 3/27/09 || 0.28841 || 18.38 ||
 * 08:3-13 || SE || Termination || 3/27/09 || 0.22005 || 24.09 ||
 * 08:3-19 || SE || Termination || 3/27/09 || 0.32172 || 16.47 ||
 * 08:3-21 || SE || Termination || 3/27/09 || 0.39619 || 13.38 ||
 * 08:3-24 || SE || Termination || 3/31/09 || 0.23793 || 22.28 ||
 * ||  ||   ||   ||   || 110.05 ||
 * 08:3-24 || SE || Termination || 3/31/09 || 0.23793 || 22.28 ||
 * ||  ||   ||   ||   || 110.05 ||

NOTE: The only change made from Sam's original pooling was exchanging sample 08:4-18 for samples 08:4-10 because there was not enough RNA to pool 5ug of 08:4-18 from the remaining stock.

Final nanodrop specs for pooled abalone total RNA:


 * Sample ID || User ID || Date || Time || ng/ul || A260 || A280 || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || 340 raw ||
 * SC total RNA pool || Default || 3/17/11 || 3:39 PM || 305.42 || 7.635 || 3.884 || 1.97 || 1.94 || 40 || 230 || 3.93 || 0.405 ||
 * SE total RNA pool || Default || 3/17/11 || 3:40 PM || 256.12 || 6.403 || 3.119 || 2.05 || 2.16 || 40 || 230 || 2.971 || 0.159 ||

Designing new hsp70 primers to Boutet et al. 2003 sequence.
 * March 16, 2011 **

Notes: Why am I doing this? The other primers I designed were to an unpublished hsp70 sequence in genebank that does not align well with the Boutet sequence. May not be measuing the gene that everyone calls hsp70 in C.gigas

Design Features: Aligned sequences for AJ305315 coding sequence and cDNA (hsc70) and AJ318882(hsp70) Note: hsp70 has no introns but sequence is extremely similar to hsc70 cDNA 1. Forward primer spans intron/exon boundary for hsc but to the hsp sequence. It is theoretically impossible to design to inton/exon boundary to hsp70 because it does not have exons. 2. The most 3' nucleotide of the forward primer is a mismatch with the hsc sequenc. 3. There are 3 basepair mismatches in the reverse primer to the hsc sequence. This represents one of the highest concentrations of basepair mismatches between the hsc and hsp sequences.

New Cgigas hsp70 primer test and Cgigas Cu/Vt hsp70 qPCR data For hsp70 normalization and graph visit the [|google doc]
 * February 10, 2011**

Testing new hsp70 C.gigas primers for detection of DNA carryover.

Used 4ng of RNA as template in a qPCR rxn. This is equivalent to the amount of RNA carried through the RT-qPCR process in which DNA carryover would be detected. These primers were designed to span Intron/Exon boundaries so there should be no genomic amplification.

I used RNA from the Cgigas Cu/Vt experiment in which I have previously detected significant amounts of DNA carryover using 18s primers.

Ran a standard 25ul qPCR rxn in 96 well plates

Results: [|RawData] Success! Absolutely no amplification of genomic DNA! qPCR amplification plot: Not the most interesting graph but still exciting!

Step 2: Rerun cDNA from Cgigas Cu/Vt experiment using new hsp70 primers. Used same reaction condition as January 21, 2011

Results: [|Raw Data] Melt curve looks a little fat...not sure what to make of that right now...

Ran qPCR for Cgigas 18s on cDNA samples from January 20, 2011. qPCR conditions were repeated from January 21, 2011 experiments.
 * January 27, 2011 **

For raw 18s qPCR data click [|here]

Results and Analysis: 1. Raw 18s data looks more consistent compared to ef1a, however there are still a few samples that appear to be "induced" from the Cu treatment. 18s "expression" data hsp70 "expression" data

2. Relative expression values were caluclated using the following formula: 1/(1+enzymatic efficiency)^Ct where the enzymatic efficiency was calculated from the standard curve generated by serially diluting a cDNA stock. hsp70 values were then divided by 18s to normalize the expression data. Check out my graph!

Testing new qPCR housekeeping gene primers. I will be running a dilution curve in duplicate for each of the 5 primer pairs using cDNA from the Cu/Vt challenge experiment.
 * January 26, 2011 **

Master Mix:


 * Reagents || 1X || 17X ||
 * Immomix || 12.50 || 212.50 ||
 * Syto13 || 1.00 || 17.00 ||
 * F Primer (10uM) || 1.25 || 21.25 ||
 * R Primer (10uM) || 1.25 || 21.25 ||
 * Water || 8.00 || 136.00 ||

Note: master mix volumes are the same for all 5 primer pairs.

Results: Everything looks pretty good. All standard curves are nice and linear and melting curves show single products except for the 28S assay, which, oddly enough, was the only assay that we did not design and came directly from a previously published study. With some tweaking of Tm, the 28s assay might be alright, but for now I will just go with 18s.

Designed new housekeeping primers for C.gigas (with Sam's help) Primers Ordered: 18s 28s ARP Actin GADPH
 * January 24, 2011 **

qPCR analysis of ef1a, hsp70, metallothionine, and copper oxidase from cDNA samples made on January 20, 2011.
 * January 21, 2011 **

qPCR mastermix Note: All volumes are reported in microliters. Volumes for mastermix are the same for all four primer pairs.
 * Reagents || 1xMix || 82xMix ||
 * Immomix || 12.5 || 1025 ||
 * Syto13 || 1 || 82 ||
 * F Primer (10uM) || 1.25 || 102.5 ||
 * R Primer (10uM) || 1.25 || 102.5 ||
 * Water || 8 || 656 ||

For a copy of the plate map click [|here].

Results: [|ef1a], [|hsp70], [|metallothionine], [|copper oxidase] 1. hsp70 and ef1a dilution curves and melting curves look good hsp70 ef1a

2. The metallothionine and copper oxidase melting curves look awful which probably explains why the dilution curves and efficiencies are not optimal. This data is unusable and assays for these genes need to be redesigned.

CopperOxidase Melt Curve Metallothionine melt curve



Sam's naming scheme

CuVt-E = Copper and Vibrio CuVt-C = Copper only Vt-C = No treatment Vt-E = Vibrio only

DNase and reverse transcription of C.gigas Cu/Vt RNA.
 * January 20, 2011 **

Reverse Transcription: Mixed the following amounts of RNA and Water to make 1ug RNA in 9ul of water.


 * Sample ID || ng/ul || ul RNA || ul water ||
 * BBC Vt-C 1 || 157.26 || 6.36 || 2.64 ||
 * BBC Vt-C 2 || 174.31 || 5.74 || 3.26 ||
 * BBC Vt-C 3 || 172.09 || 5.81 || 3.19 ||
 * BBC Vt-C 4 || 175.17 || 5.71 || 3.29 ||
 * BBC Vt-C 5 || 157.86 || 6.33 || 2.67 ||
 * BBC Vt-C 6 || 166.85 || 5.99 || 3.01 ||
 * BBC Vt-C 7 || 173.25 || 5.77 || 3.23 ||
 * BBC Vt-E 1 || 157.34 || 6.36 || 2.64 ||
 * BBC Vt-E 2 || 150.43 || 6.65 || 2.35 ||
 * BBC Vt-E 3 || 172 || 5.81 || 3.19 ||
 * BBC Vt-E 4 || 164.33 || 6.09 || 2.91 ||
 * BBC Vt-E 5 || 155.94 || 6.41 || 2.59 ||
 * BBC Vt-E 6 || 176.26 || 5.67 || 3.33 ||
 * BBC Vt-E 7 || 175.04 || 5.71 || 3.29 ||
 * BBC Vt-E 8 || 171.14 || 5.84 || 3.16 ||
 * BBC CuVt-C 1 || 216.32 || 4.62 || 4.38 ||
 * BBC CuVt-C 2 || 152.54 || 6.56 || 2.44 ||
 * BBC CuVt-C 3 || 191.14 || 5.23 || 3.77 ||
 * BBC CuVt-C 4 || 158.52 || 6.31 || 2.69 ||
 * BBC CuVt-C 5 || 177.49 || 5.63 || 3.37 ||
 * BBC CuVt-C 6 || 156.51 || 6.39 || 2.61 ||
 * BBC CuVt-C 7 || 167.94 || 5.95 || 3.05 ||
 * BBC CuVt-C 8 || 156.93 || 6.37 || 2.63 ||
 * BBC CuVt-E 1 || 197.47 || 5.06 || 3.94 ||
 * BBC CuVt-E 2 || 151.45 || 6.60 || 2.40 ||
 * BBC CuVt-E 3 || 146.55 || 6.82 || 2.18 ||
 * BBC CuVt-E 5 || 155.74 || 6.42 || 2.58 ||
 * BBC CuVt-E 6 || 151.38 || 6.61 || 2.39 ||
 * BBC CuVt-E 7 || 154.16 || 6.49 || 2.51 ||
 * BBC CuVt-E 8 || 163.46 || 6.12 || 2.88 ||

Added 1ul Oligo DT and preannealed at 70degreesC for 5min. Placed samples on Ice immediately. Combined the following reagents in a master mix and added 15ul of mix to each sample.


 * RT Rxn || 1x || 33x ||
 * 5x MMLV Buffer || 5 || 165 ||
 * dNTPs || 5 || 165 ||
 * MMLV || 1 || 33 ||
 * Water || 4 || 132 ||

Incubated at 42 degrees for 1hr.

DNase treatment of Cgigas RNA from Cu/Vt experiment: Follow manufacturer's recommended protocol. Combined the following volumes of RNA and water for a total of 10ug of nucleic acid/rxn


 * Sample ID || ng/ul || ul RNA || ul Water ||
 * BBC Vt-C 1 || 690.14 || 14.49 || 30.51 ||
 * BBC Vt-C 2 || 1034.58 || 9.67 || 35.33 ||
 * BBC Vt-C 3 || 364.38 || 27.44 || 17.56 ||
 * BBC Vt-C 4 || 393.19 || 25.43 || 19.57 ||
 * BBC Vt-C 5 || 747.36 || 13.38 || 31.62 ||
 * BBC Vt-C 6 || 724.79 || 13.80 || 31.20 ||
 * BBC Vt-C 7 || 859.95 || 11.63 || 33.37 ||
 * BBC CuVt-E 1 || 1241.48 || 8.05 || 36.95 ||
 * BBC CuVt-E 2 || 710.51 || 14.07 || 30.93 ||
 * BBC CuVt-E 3 || 556.61 || 17.97 || 27.03 ||
 * BBC CuVt-E 5 || 685.71 || 14.58 || 30.42 ||
 * BBC CuVt-E 6 || 886.43 || 11.28 || 33.72 ||
 * BBC CuVt-E 7 || 681.74 || 14.67 || 30.33 ||
 * BBC CuVt-E 8 || 935.94 || 10.68 || 34.32 ||
 * BBC Vt-E 1 || 835.09 || 11.97 || 33.03 ||
 * BBC Vt-E 2 || 715.66 || 13.97 || 31.03 ||
 * BBC Vt-E 3 || 926.94 || 10.79 || 34.21 ||
 * BBC Vt-E 4 || 839.53 || 11.91 || 33.09 ||
 * BBC Vt-E 5 || 644.07 || 15.53 || 29.47 ||
 * BBC Vt-E 6 || 980.71 || 10.20 || 34.80 ||
 * BBC Vt-E 7 || 944.86 || 10.58 || 34.42 ||
 * BBC Vt-E 8 || 402.64 || 24.84 || 20.16 ||
 * BBC CuVt-C 1 || 654.15 || 15.29 || 29.71 ||
 * BBC CuVt-C 2 || 638.36 || 15.67 || 29.33 ||
 * BBC CuVt-C 2 || 641.84 || 15.58 || 29.42 ||
 * BBC CuVt-C 3 || 794.99 || 12.58 || 32.42 ||
 * BBC CuVt-C 4 || 668.69 || 14.95 || 30.05 ||
 * BBC CuVt-C 5 || 812.48 || 12.31 || 32.69 ||
 * BBC CuVt-C 6 || 797.99 || 12.53 || 32.47 ||
 * BBC CuVt-C 7 || 775.26 || 12.90 || 32.10 ||
 * BBC CuVt-C 8 || 1066.56 || 9.38 || 35.62 ||
 * BBC CuVt-C 8 || 1066.3 || 9.38 || 35.62 ||
 * BBC CuVt-C 8 || 801.9 || 12.47 || 32.53 ||

RNA extraction of BBC Vt-E RNA from C.gigas CuVt experiment. BBC Vt-E = Big Beef Creek oysters challenged with Vt only.
 * January 19, 2011 **

RNA was extracted using 1ml TriReagent following manufacturer's recommended protocol. RNA was resuspended in ~250ul of water and stored at -80.


 * = Sample ID ||= User ID ||= Date ||= Time ||= ng/ul ||= A260 ||= A280 ||= 260/280 ||= 260/230 ||= Constant ||= Cursor Pos. ||= Cursor abs. ||= 340 raw ||
 * BBC Vt-E 1 || Default || 1/19/11 || 2:58 PM || 835.09 || 20.877 || 10.589 || 1.97 || 1.87 || 40 || 230 || 11.17 || 0.637 ||
 * BBC Vt-E 2 || Default || 1/19/11 || 3:00 PM || 715.66 || 17.892 || 9.122 || 1.96 || 1.57 || 40 || 230 || 11.392 || 0.806 ||
 * BBC Vt-E 3 || Default || 1/19/11 || 3:02 PM || 926.94 || 23.173 || 11.603 || 2 || 1.59 || 40 || 230 || 14.582 || 0.866 ||
 * BBC Vt-E 4 || Default || 1/19/11 || 3:04 PM || 839.53 || 20.988 || 10.573 || 1.99 || 1.74 || 40 || 230 || 12.035 || 0.749 ||
 * BBC Vt-E 5 || Default || 1/19/11 || 3:04 PM || 644.07 || 16.102 || 8.341 || 1.93 || 2.24 || 40 || 230 || 7.18 || 0.219 ||
 * BBC Vt-E 6 || Default || 1/19/11 || 3:05 PM || 980.71 || 24.518 || 12.396 || 1.98 || 1.81 || 40 || 230 || 13.554 || 0.886 ||
 * BBC Vt-E 7 || Default || 1/19/11 || 3:07 PM || 944.86 || 23.622 || 11.903 || 1.98 || 1.5 || 40 || 230 || 15.74 || 1.118 ||
 * BBC Vt-E 8 || Default || 1/19/11 || 3:08 PM || 402.64 || 10.066 || 5.413 || 1.86 || 2.01 || 40 || 230 || 5.011 || 0.317 ||


 * January 18, 2010 **

RNA extraction of BBC CuVt-C RNA from C.gigas CuVt experiment. BBC CuVt-C = Big Beef Creek oysters challenged with Cu only.

RNA was extracted using 1ml TriReagent following manufacturer's recommended protocol. RNA was resuspended in ~250ul of water and stored at -80.


 * Sample ID || User ID || Date || Time || ng/ul || A260 || A280 || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || 340 raw ||
 * BBC CuVt-C 1 || Default || 1/18/11 || 2:08 PM || 654.15 || 16.354 || 8.125 || 2.01 || 1.13 || 40 || 230 || 14.485 || 1.054 ||
 * BBC CuVt-C 2 || Default || 1/18/11 || 2:09 PM || 641.84 || 16.046 || 7.959 || 2.02 || 1.45 || 40 || 230 || 11.097 || 0.778 ||
 * BBC CuVt-C 3 || Default || 1/18/11 || 2:10 PM || 794.99 || 19.875 || 9.481 || 2.1 || 1.94 || 40 || 230 || 10.268 || 0.532 ||
 * BBC CuVt-C 4 || Default || 1/18/11 || 2:11 PM || 668.69 || 16.717 || 8.397 || 1.99 || 1.87 || 40 || 230 || 8.938 || 0.601 ||
 * BBC CuVt-C 5 || Default || 1/18/11 || 2:11 PM || 812.48 || 20.312 || 10.107 || 2.01 || 2.14 || 40 || 230 || 9.492 || 0.375 ||
 * BBC CuVt-C 6 || Default || 1/18/11 || 2:12 PM || 797.99 || 19.95 || 9.956 || 2 || 1.91 || 40 || 230 || 10.468 || 0.511 ||
 * BBC CuVt-C 7 || Default || 1/18/11 || 2:12 PM || 775.26 || 19.382 || 9.859 || 1.97 || 1.97 || 40 || 230 || 9.826 || 0.505 ||
 * BBC CuVt-C 8 || Default || 1/18/11 || 2:15 PM || 801.9 || 20.047 || 9.851 || 2.04 || 1.49 || 40 || 230 || 13.442 || 0.778 ||


 * January 14, 2011**

RNA extraction of BBC Vt-C and BBC CuVt-E RNA from C.gigas CuVt experiment. BBC Vt-C = Big Beef Creek control oysters. Received no treatment. BBC CuVt-E = Big Beef Creed oysters challenged with Cu and Vt.

RNA was extracted using 1ml TriReagent following manufacturer's recommended protocol. RNA was resuspended in ~250ul of water and stored at -80.


 * Sample ID || User ID || Date || Time || ng/ul || A260 || A280 || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || 340 raw ||
 * BBC Vt-C 1 || Default || 1/14/11 || 5:20 PM || 690.14 || 17.254 || 8.676 || 1.99 || 1.72 || 40 || 230 || 10.055 || 0.753 ||
 * BBC Vt-C 2 || Default || 1/14/11 || 5:21 PM || 1034.58 || 25.865 || 12.733 || 2.03 || 1.69 || 40 || 230 || 15.341 || 0.972 ||
 * BBC Vt-C 3 || Default || 1/14/11 || 5:21 PM || 364.38 || 9.11 || 4.769 || 1.91 || 2.2 || 40 || 230 || 4.134 || 0.24 ||
 * BBC Vt-C 4 || Default || 1/14/11 || 5:22 PM || 393.19 || 9.83 || 5.146 || 1.91 || 2.04 || 40 || 230 || 4.817 || 0.325 ||
 * BBC Vt-C 5 || Default || 1/14/11 || 5:22 PM || 747.36 || 18.684 || 9.283 || 2.01 || 2.05 || 40 || 230 || 9.118 || 0.52 ||
 * BBC Vt-C 6 || Default || 1/14/11 || 5:23 PM || 724.79 || 18.12 || 9.078 || 2 || 2.04 || 40 || 230 || 8.892 || 0.539 ||
 * BBC Vt-C 7 || Default || 1/14/11 || 5:23 PM || 859.95 || 21.499 || 10.677 || 2.01 || 1.43 || 40 || 230 || 15.078 || 1.152 ||
 * BBC CuVt-E 1 || Default || 1/14/11 || 5:24 PM || 1241.48 || 31.037 || 15.306 || 2.03 || 1.96 || 40 || 230 || 15.852 || 0.769 ||
 * BBC CuVt-E 2 || Default || 1/14/11 || 5:24 PM || 710.51 || 17.763 || 9.009 || 1.97 || 2.18 || 40 || 230 || 8.155 || 0.362 ||
 * BBC CuVt-E 3 || Default || 1/14/11 || 5:25 PM || 556.61 || 13.915 || 7.071 || 1.97 || 1.85 || 40 || 230 || 7.514 || 0.492 ||
 * BBC CuVt-E 5 || Default || 1/14/11 || 5:25 PM || 685.71 || 17.143 || 8.687 || 1.97 || 2.26 || 40 || 230 || 7.585 || 0.373 ||
 * BBC CuVt-E 6 || Default || 1/14/11 || 5:26 PM || 886.43 || 22.161 || 11.035 || 2.01 || 1.8 || 40 || 230 || 12.301 || 0.911 ||
 * BBC CuVt-E 7 || Default || 1/14/11 || 5:26 PM || 681.74 || 17.044 || 8.658 || 1.97 || 2.24 || 40 || 230 || 7.601 || 0.304 ||
 * BBC CuVt-E 8 || Default || 1/14/11 || 5:28 PM || 935.94 || 23.399 || 11.442 || 2.04 || 1.67 || 40 || 230 || 14.048 || 0.935 ||

qPCR assays for Actin, Serine Protease Inhibitor, and Big Defensin using cDNA from January 11, 2011
 * January 12, 2011**

diluted cDNA 1:5 and loaded 1ul/rxn Ran a standard curve of 0, 1:5, 1:25, 1:125, and 1:625 dilutions of pooled cDNA from each sample.

Results: 1. Serine Protease Inhibitor was not detectable in any of the samples. 2. Amplification of other genes as sporadic. Only Seawater, ATTC, and MA4 hemocytes expressed the Actin housekeeping gene. 3. Expression levels were not strong enough to generate a robust standard curve. The following equation was applied to calculate relative expression values. =10^(-(0.3012*//Ct//)+11.434) ATTC and Seawater Big Defensin expression values were then normalized to actin.The corresponding graph is below.
 * Master Mix || 1x || 25x ||
 * Immunomix || 12.5 || 312.5 ||
 * Syto 13 || 1 || 25 ||
 * F Primer || 1.25 || 31.25 ||
 * R Primer || 1.25 || 31.25 ||
 * Water || 8 || 200 ||

4. BigDef qPCR assay is also not optimized. See melting curve below for further explanation... Reverse Transcribed DNased RNA from January 10, 2011 Used 1ug RNA/Rxn
 * January 11, 2011 **


 * Sample ID || ng/ul || ul RNA || ul Water ||
 * SEA 1 || 6.19 || 9.00 || 0.00 ||
 * SEA 2 || 121.78 || 8.21 || 0.79 ||
 * SEA 3 || 141.12 || 7.09 || 1.91 ||
 * ATTC 1 || 42.91 || 9.00 || 0.00 ||
 * ATTC 2 || 118.09 || 8.47 || 0.53 ||
 * ATTC 3 || 268.66 || 3.72 || 5.28 ||
 * S-1 1 || 25.77 || 9.00 || 0.00 ||
 * S-1 2 || 131.7 || 7.59 || 1.41 ||
 * S-1 3 || 135.44 || 7.38 || 1.62 ||
 * TD8-8 1 || 11.25 || 9.00 || 0.00 ||
 * TD8-8 2 || 128.26 || 7.80 || 1.20 ||
 * TD8-8 3 || 181.76 || 5.50 || 3.50 ||
 * BX-1 hemocyte || 24.36 || 9.00 || 0.00 ||
 * MA4 hemocyte || 73.64 || 9.00 || 0.00 ||
 * 1ul Oligo dT ||  ||   ||   ||
 * || 1x || 16x ||  ||
 * MMLV 5x MM || 5 || 80 ||  ||
 * dNTP || 5 || 80 ||  ||
 * RT || 1 || 16 ||  ||
 * Water || 4 || 64 ||  ||
 * Water || 4 || 64 ||  ||

DNase treatment of QPX "tissue immersion" samples. Continued from December 15, 2010.
 * January 10, 2011**

Treated ~8ug of RNA (unless I did not have that much, in which case I treated everything I had for that sample) with DNase to remove possible DNA contamination. This is in accordance with the recommended maximum of 10ug RNA/1ul DNase enzyme.

Followed manufacturers recommended protocol.

Results from Nanodrop


 * Sample ID || Date || ng/ul || A260 || A280 || 260/280 || 260/230 ||
 * SEA 1 || 1/10/11 || 6.19 || 0.155 || 0.107 || 1.45 || 0.13 ||
 * SEA 2 || 1/10/11 || 121.78 || 3.044 || 1.536 || 1.98 || 1.55 ||
 * SEA 3 || 1/10/11 || 141.12 || 3.528 || 1.729 || 2.04 || 1.53 ||
 * ATTC 1 || 1/10/11 || 42.91 || 1.073 || 0.53 || 2.03 || 0.86 ||
 * ATTC 2 || 1/10/11 || 118.09 || 2.952 || 1.512 || 1.95 || 1.01 ||
 * ATTC 3 || 1/10/11 || 268.66 || 6.717 || 3.326 || 2.02 || 1.29 ||
 * S-1 1 || 1/10/11 || 25.77 || 0.644 || 0.385 || 1.67 || 0.37 ||
 * S-1 2 || 1/10/11 || 131.7 || 3.292 || 1.641 || 2.01 || 1.41 ||
 * S-1 3 || 1/10/11 || 135.44 || 3.386 || 1.668 || 2.03 || 1.42 ||
 * TD8-8 1 || 1/10/11 || 11.25 || 0.281 || 0.174 || 1.62 || 0.23 ||
 * TD8-8 2 || 1/10/11 || 128.26 || 3.207 || 1.619 || 1.98 || 1.38 ||
 * TD8-8 3 || 1/10/11 || 181.76 || 4.544 || 2.26 || 2.01 || 1.38 ||
 * BX-1 hemocyte || 1/10/11 || 24.36 || 0.609 || 0.367 || 1.66 || 0.3 ||
 * MA4 hemocyte || 1/10/11 || 73.64 || 1.841 || 0.983 || 1.87 || 0.74 ||

Results: Samples look pretty good. Samples #1 from all treatments have very low RNA yields which was to be expected since these represent extractions of QPX floating around in seawater while the other samples are RNA extracted from Hard Clam gill tissue.

**January 7, 2011** Testing RNA from the class experiment (exposing C.gigas to Cu and Vt) for DNA contamination.

Loaded 16ng of RNA (equivalent to the amount RNA "contamination" carried through from the RT reaction to the qPCR) Calculation: 2ugRNA/RT reaction. RT reaction diluted to 250ul. 2ugRNA/250ul = .008. 2ul of RT reaction (.008ugRNA/ul) loaded in qPCR = 16 total ng of total RNA in RT reaction.

Results: CONTAMINATION!



What do to now? There are bascially three options... 1. ReDNase the RNA samples. According to the class notes, the samples were previously DNAsed. Repeating this might compromise the integrity of the RNA samples. 2. Extract more RNA from extra tissue samples. This is the safest route to go, however, it is also the most time consuming and costly. 3. Drop the experiment altogether. Easiest solution but perhaps also most costly since we have already put in all of the time and effort to treat the oysters, collect the samples, and get the hsp70 protein data.

I'm leaning heavily towards option #2 but will have to talk with Steven about this before moving forward.

Todays the final push to produce some data before the holidays!
 * December 23, 2010**

I'll be running some qPCR assays on the hard clam cDNA samples from yesterday. Because I'm short on plate space I will only be doing qPCR assays from a single sampling group; BX, MA, and FL.

Actin: normalizing gene Serine protease inhibitor: the assay that I've been playing with this quarter. Preliminary data from Steven suggests that I could have interesting results in the FL samples.
 * Assays Run**:

[|Click here for Actin qPCR data file.]

[|Click here for SPI qPCR data file.]

Actin Standard Curve

SPI Standard Curve



Results!

Today I will be DNasing and RTing all of the samples that I will be using for gene expression for the Hard Clam QPX project. Nothing too special to report, just the final RNA concentrations of the DNased samples...
 * December 22, 2010 **


 * Master Mix || 1x || 50 ||
 * MMLV 5x Buffer || 5 || 250 ||
 * dNTP || 5 || 250 ||
 * RT || 1 || 50 ||
 * Water || 4 || 200 ||

The Table below has the volumes of RNA and water used in the RT reaction to make 1ug total RNA/Rxn
 * Sample ID || ng/ul || ul RNA || ul H20 ||
 * CA 4 || 162.95 || 6.1 || 2.9 ||
 * CA 5 DNase || 173.65 || 5.8 || 3.2 ||
 * DNase CA 6 || 167.86 || 6.0 || 3.0 ||
 * DNase CA 7 || 172.86 || 5.8 || 3.2 ||
 * DNase CA 8 || 95.62 || 9.0 || 0.0 ||
 * DNase CA 9 || 159.47 || 6.3 || 2.7 ||
 * DNase CA 10 || 84.97 || 9.0 || 0.0 ||
 * DNase MA 4 || 151.54 || 6.6 || 2.4 ||
 * DNase MA 5 || 147.08 || 6.8 || 2.2 ||
 * DNase MA 6 || 221.32 || 4.5 || 4.5 ||
 * DNase MA 7 || 112.1 || 9.0 || 0.0 ||
 * DNase MA 8 || 106.57 || 9.0 || 0.0 ||
 * DNase MA 9 || 150.03 || 6.7 || 2.3 ||
 * DNase MA 10 || 158.45 || 6.3 || 2.7 ||
 * DNase BX 1-3 || 187.94 || 5.3 || 3.7 ||
 * DNase BX 1-4 || 157.42 || 6.4 || 2.6 ||
 * DNase BX 2-2 || 92.9 || 9.0 || 0.0 ||
 * DNase BX 2-3 || 155.63 || 6.4 || 2.6 ||
 * DNase BX 3-2 || 175.77 || 5.7 || 3.3 ||
 * DNase BX 3-3 || 168.07 || 5.9 || 3.1 ||
 * DNase BX 4-1 || 170.07 || 5.9 || 3.1 ||
 * DNase BX 4-2 || 138.08 || 7.2 || 1.8 ||
 * DNase MA 1-3 || 179.59 || 5.6 || 3.4 ||
 * DNase MA 1-5 || 138.11 || 7.2 || 1.8 ||
 * DNase MA 2-4 || 177.13 || 5.6 || 3.4 ||
 * DNase MA 2-5 || 163.88 || 6.1 || 2.9 ||
 * DNase MA 3-3 || 151.42 || 6.6 || 2.4 ||
 * DNase MA 3-4 || 165.03 || 6.1 || 2.9 ||
 * DNase MA 4-3 || 157.76 || 6.3 || 2.7 ||
 * DNase MA 4-4 || 123.83 || 8.1 || 0.9 ||
 * Max 4 DNase || 172.32 || 5.8 || 3.2 ||
 * Max 5 DNase || 73.09 || 9.0 || 0.0 ||
 * Max 6 DNase || 44.17 || 9.0 || 0.0 ||
 * Max 7 DNase || 47.22 || 9.0 || 0.0 ||
 * Max 8 DNase || 45.37 || 9.0 || 0.0 ||
 * Max 9 DNase || 111.74 || 9.0 || 0.0 ||
 * Max 10 DNase || 73.46 || 9.0 || 0.0 ||

Useing Ambion's DNA-free Turbo kit... Combined 5ul of 10x reaction buffer with the RNA and Water volumes in the table below. Added 1ul DNase enzyme and incubated at 37 degrees C for 30min. Added. 5.5ul inactivation reagent. Incubate RT 2min. Spin 10k 1.5min. Transfer supt to a new tube and spec.
 * DNase Treatment**

Note: MAX and FL samples were previously DNased here and here.


 * Sample ID || User ID || Date || Time || ng/ul || A260 || A280 || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || 340 raw ||
 * Dnase CA 4 || Default || 12/22/10 || 12:14 PM || 162.95 || 4.074 || 2.08 || 1.96 || 1.43 || 40 || 230 || 2.847 || 0.035 ||
 * Dnase CA 5 || Default || 12/22/10 || 12:15 PM || 173.65 || 4.341 || 2.24 || 1.94 || 1.56 || 40 || 230 || 2.779 || -0.003 ||
 * DNase CA 6 || Default || 12/22/10 || 12:16 PM || 167.86 || 4.197 || 2.142 || 1.96 || 1.43 || 40 || 230 || 2.939 || 0.024 ||
 * DNase CA 7 || Default || 12/22/10 || 12:16 PM || 172.86 || 4.322 || 2.216 || 1.95 || 1.46 || 40 || 230 || 2.96 || -0.01 ||
 * DNase CA 8 || Default || 12/22/10 || 12:17 PM || 95.62 || 2.391 || 1.243 || 1.92 || 1.19 || 40 || 230 || 2.006 || 0.062 ||
 * DNase CA 9 || Default || 12/22/10 || 12:17 PM || 159.47 || 3.987 || 2.04 || 1.95 || 1.4 || 40 || 230 || 2.85 || -0.007 ||
 * DNase CA 10 || Default || 12/22/10 || 12:18 PM || 84.97 || 2.124 || 1.101 || 1.93 || 1.45 || 40 || 230 || 1.468 || 0.001 ||
 * DNase MA 4 || Default || 12/22/10 || 12:18 PM || 151.54 || 3.788 || 1.962 || 1.93 || 1.66 || 40 || 230 || 2.284 || 0.021 ||
 * DNase MA 5 || Default || 12/22/10 || 12:19 PM || 147.08 || 3.677 || 1.948 || 1.89 || 1.85 || 40 || 230 || 1.988 || -0.003 ||
 * DNase MA 6 || Default || 12/22/10 || 12:19 PM || 221.32 || 5.533 || 2.867 || 1.93 || 1.88 || 40 || 230 || 2.938 || 0.026 ||
 * DNase MA 7 || Default || 12/22/10 || 12:20 PM || 112.1 || 2.803 || 1.469 || 1.91 || 1.35 || 40 || 230 || 2.073 || 0.139 ||
 * DNase MA 8 || Default || 12/22/10 || 12:20 PM || 106.57 || 2.664 || 1.394 || 1.91 || 1.51 || 40 || 230 || 1.761 || 0.029 ||
 * DNase MA 9 || Default || 12/22/10 || 12:21 PM || 150.03 || 3.751 || 1.956 || 1.92 || 1.9 || 40 || 230 || 1.972 || -0.007 ||
 * DNase MA 10 || Default || 12/22/10 || 12:21 PM || 158.45 || 3.961 || 2.065 || 1.92 || 1.82 || 40 || 230 || 2.179 || 0.011 ||
 * DNase BX 1-3 || Default || 12/22/10 || 12:22 PM || 187.94 || 4.698 || 2.481 || 1.89 || 1.89 || 40 || 230 || 2.482 || -0.015 ||
 * DNase BX 1-4 || Default || 12/22/10 || 12:23 PM || 157.42 || 3.936 || 1.999 || 1.97 || 1.83 || 40 || 230 || 2.151 || -0.004 ||
 * DNase BX 2-2 || Default || 12/22/10 || 12:23 PM || 92.9 || 2.322 || 1.202 || 1.93 || 1.62 || 40 || 230 || 1.431 || -0.018 ||
 * DNase BX 2-3 || Default || 12/22/10 || 12:24 PM || 155.63 || 3.891 || 2.01 || 1.94 || 1.85 || 40 || 230 || 2.104 || -0.009 ||
 * DNase BX 3-2 || Default || 12/22/10 || 12:24 PM || 175.77 || 4.394 || 2.363 || 1.86 || 1.85 || 40 || 230 || 2.37 || -0.009 ||
 * DNase BX 3-3 || Default || 12/22/10 || 12:25 PM || 168.07 || 4.202 || 2.227 || 1.89 || 1.87 || 40 || 230 || 2.249 || -0.015 ||
 * DNase BX 4-1 || Default || 12/22/10 || 12:25 PM || 170.07 || 4.252 || 2.262 || 1.88 || 1.42 || 40 || 230 || 2.989 || 0.258 ||
 * DNase BX 4-2 || Default || 12/22/10 || 12:26 PM || 138.08 || 3.452 || 1.847 || 1.87 || 1.41 || 40 || 230 || 2.446 || 0.203 ||
 * DNase MA 1-3 || Default || 12/22/10 || 12:26 PM || 179.59 || 4.49 || 2.306 || 1.95 || 1.85 || 40 || 230 || 2.431 || -0.02 ||
 * DNase MA 1-5 || Default || 12/22/10 || 12:27 PM || 138.11 || 3.453 || 1.818 || 1.9 || 1.84 || 40 || 230 || 1.873 || -0.01 ||
 * DNase MA 2-4 || Default || 12/22/10 || 12:27 PM || 177.13 || 4.428 || 2.27 || 1.95 || 1.75 || 40 || 230 || 2.526 || -0.007 ||
 * DNase MA 2-5 || Default || 12/22/10 || 12:28 PM || 163.88 || 4.097 || 2.089 || 1.96 || 1.79 || 40 || 230 || 2.294 || 0.001 ||
 * DNase MA 3-3 || Default || 12/22/10 || 12:28 PM || 151.42 || 3.785 || 1.939 || 1.95 || 1.8 || 40 || 230 || 2.105 || -0.009 ||
 * DNase MA 3-4 || Default || 12/22/10 || 12:29 PM || 165.03 || 4.126 || 2.142 || 1.93 || 1.77 || 40 || 230 || 2.337 || -0.012 ||
 * DNase MA 4-3 || Default || 12/22/10 || 12:29 PM || 157.76 || 3.944 || 2.028 || 1.95 || 1.86 || 40 || 230 || 2.124 || -0.017 ||
 * DNase MA 4-4 || Default || 12/22/10 || 12:30 PM || 123.83 || 3.096 || 1.67 || 1.85 || 1.82 || 40 || 230 || 1.7 || -0.015 ||

The table below has the volumes of RNA used in the DNase treatment so as not to exceel 10ug of total RNA/Rxn. The reported concentration in this table is the previously reported concentration BEFORE DNase treatment.
 * Sample ID || ng/ul || ul RNA || ul water ||
 * BX1-3 || 403.66 || 24.77 || 20.23 ||
 * BX1-4 || 260.08 || 38.45 || 6.55 ||
 * BX2-2 || 137.09 || 45.00 || 0.00 ||
 * BX2-3 || 240.71 || 41.54 || 3.46 ||
 * BX3-2 || 279.37 || 35.79 || 9.21 ||
 * BX3-3 || 266.55 || 37.52 || 7.48 ||
 * BX4-1 || 329.88 || 30.31 || 14.69 ||
 * BX4-2 || 193.62 || 45.00 || 0.00 ||
 * MA1-3 || 335.15 || 29.84 || 15.16 ||
 * MA1-5 || 176.71 || 45.00 || 0.00 ||
 * MA2-4 || 452.18 || 22.12 || 22.88 ||
 * MA2-5 || 379.45 || 26.35 || 18.65 ||
 * MA3-3 || 213.62 || 45.00 || 0.00 ||
 * MA3-4 || 246.55 || 40.56 || 4.44 ||
 * MA4-3 || 230.18 || 43.44 || 1.56 ||
 * MA4-4 || 163.11 || 45.00 || 0.00 ||
 * CA 4 || 256 || 39.06 || 5.94 ||
 * CA 5 || 259 || 38.61 || 6.39 ||
 * CA 6 || 302 || 33.11 || 11.89 ||
 * CA 7 || 224 || 44.64 || 0.36 ||
 * CA 8 || 130 || 45.00 || 0.00 ||
 * CA 9 || 207 || 45.00 || 0.00 ||
 * MA 4 || 719 || 13.91 || 31.09 ||
 * MA 5 || 708 || 14.12 || 30.88 ||
 * MA 6 || 350 || 28.57 || 16.43 ||
 * MA 7 || 160 || 45.00 || 0.00 ||
 * MA 8 || 155 || 45.00 || 0.00 ||
 * MA 9 || 661 || 15.13 || 29.87 ||
 * MA 10 || 573 || 17.45 || 27.55 ||
 * || 121 || 45.00 ||  ||

M. mercanaria QPX gill challenege experiment continued from October 26, 2010
 * December 15, 2010**

RNA extractions using TriReagent


 * Sample ID || User ID || Date || Time || ng/ul || A260 || A280 || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || 340 raw ||
 * SEA 1 || Default || 12/16/10 || 12:16 PM || 30.15 || 0.754 || 0.477 || 1.58 || 0.25 || 40 || 230 || 3.034 || 0.029 ||
 * SEA 2 || Default || 12/16/10 || 12:30 PM || 911.87 || 22.797 || 11.597 || 1.97 || 1.89 || 40 || 230 || 12.077 || 0.438 ||
 * SEA 3 || Default || 12/16/10 || 12:18 PM || 785.72 || 19.643 || 9.88 || 1.99 || 1.82 || 40 || 230 || 10.81 || 0.019 ||
 * S-1 1 || Default || 12/16/10 || 12:18 PM || 70.85 || 1.771 || 1.064 || 1.66 || 0.46 || 40 || 230 || 3.83 || 0.026 ||
 * S-1 2 || Default || 12/16/10 || 12:19 PM || 583.63 || 14.591 || 7.497 || 1.95 || 1.76 || 40 || 230 || 8.275 || 0.298 ||
 * S1-3 || Default || 12/16/10 || 12:31 PM || 788.28 || 19.707 || 9.925 || 1.99 || 1.7 || 40 || 230 || 11.615 || 0.16 ||
 * ATTC 1 || Default || 12/16/10 || 12:25 PM || 133.97 || 3.349 || 1.704 || 1.97 || 1.25 || 40 || 230 || 2.672 || -0.001 ||
 * ATTC 2 || Default || 12/16/10 || 12:21 PM || 239.9 || 5.997 || 3.177 || 1.89 || 0.98 || 40 || 230 || 6.102 || 0.066 ||
 * ATTC 3 || Default || 12/16/10 || 12:22 PM || 387.17 || 9.679 || 5.006 || 1.93 || 1.29 || 40 || 230 || 7.491 || 0.019 ||
 * TD8-81 1 || Default || 12/16/10 || 12:25 PM || 31.73 || 0.793 || 0.5 || 1.59 || 0.31 || 40 || 230 || 2.523 || -0.008 ||
 * TD8-81 2 || Default || 12/16/10 || 12:26 PM || 596.38 || 14.91 || 7.74 || 1.93 || 1.62 || 40 || 230 || 9.216 || 0.367 ||
 * TD8-81 3 || Default || 12/16/10 || 12:26 PM || 312.45 || 7.811 || 4.024 || 1.94 || 1.57 || 40 || 230 || 4.979 || 0.743 ||
 * BX1 Hemo || Default || 12/16/10 || 12:27 PM || 57.36 || 1.434 || 0.847 || 1.69 || 0.32 || 40 || 230 || 4.508 || 0.781 ||
 * MA4 Hemo || Default || 12/16/10 || 12:28 PM || 144.83 || 3.621 || 2.038 || 1.78 || 0.58 || 40 || 230 || 6.282 || 1.337 ||

Naming Scheme: SEA 1 = RNA from seawater only SEA 2 = RNA from gill tissue incubated in seawater SEA 3 = RNA from water that had the gill tissue

S-1 1 = RNA from QPX strain SP-1 in seawater only S-1 2 = RNA from Gill tissue challenged with SP-1 S-1 3 = RNA from seawater of Gill tissue challenge with SP-1

ATTC 1 = RNA from QPX strain ATTC 1 in seawater only ATTC 2 = RNA from Gill tissue Challenged with ATTC ATTC 3 = RNA from seawater of Gill tissue challenge with ATTC

TD8-81 1 = RNA from QPX strain TD8-81 in seawater only TD8-81 2 = RNA from Gill tissue Challenged with TD8-81 TD8-81 3 = RNA from seawater of Gill tissue challenged with TD8-81

BX1 = Plated hemocyte RNA from clam BX1 MA4 = Plated hemocyte RNA from clam MA4

Testing Serine Protease Inhibitor qPCR primers on FL samples from December 13, 2010 The SPI primers were ones that I had made in class and were previously tested on a single sample. Results were a little inconclusive. While I sa expression, it was very low and did not give me an indication of how the assay was working. In this experiment I am going to run replicates of a dilution curve to look for amplification levels in FL samples, optimal cDNA dilutions, CV between replicates, and efficiency of assay (aka are the dilutions linear).
 * December 14, 2010 **

Dilutions = 0, 1:5, 1:10, 1:50, 1:100, 1:500

Plate Map/Results


 * Well || Fluor || Content || Sample || C(t) ||
 * A01 || FAM || Unkn || 1 || 33.95 ||
 * A02 || FAM || Unkn || .2 || 36.49 ||
 * A03 || FAM || Unkn || .1 || 37.59 ||
 * A04 || FAM || Unkn || .02 || 38.91 ||
 * A05 || FAM || Unkn || .01 ||  ||
 * A06 || FAM || Unkn || .002 ||  ||
 * A07 || FAM || Unkn || NTC ||  ||
 * A08 || FAM || Unkn || Blank ||  ||
 * A09 || FAM || Unkn || Blank ||  ||
 * A10 || FAM || Unkn || Blank ||  ||
 * A11 || FAM || Unkn || Blank ||  ||
 * A12 || FAM || Unkn || Blank ||  ||
 * B01 || FAM || Unkn || 1 || 33.80 ||
 * B02 || FAM || Unkn || .2 || 36.83 ||
 * B03 || FAM || Unkn || .1 || 38.09 ||
 * B04 || FAM || Unkn || .02 || 39.27 ||
 * B05 || FAM || Unkn || .01 ||  ||
 * B06 || FAM || Unkn || .002 ||  ||
 * B07 || FAM || Unkn || NTC ||  ||
 * B08 || FAM || Unkn || Blank ||  ||
 * B09 || FAM || Unkn || Blank ||  ||
 * B10 || FAM || Unkn || Blank ||  ||
 * B11 || FAM || Unkn || Blank ||  ||
 * B12 || FAM || Unkn || Blank ||  ||



Conclusions: 1. Assay looks pretty good. R square of 0.97 is pretty tight although it would be nice if it was closer to .99 but the Ct values are getting pretty by the 1:10 dilution that this may not be possible for this set of concentrations. 2. SPI seems to be expressed in the samples, albeit at fairly low levels. 3. SPI was detected at 1:50 but was outside the linear range of the assay. 1:100 and 1:500 dilutions had no signal. 4. For future experiments I would run cDNA samples somewhere between their most concentrated form and a 1:5 dilution...maybe 1:2 is best to save on reagents.

WooHoo! back in the lab! and just shy under a month too!
 * December 13, 2010 **

RT reactions on FL samples DNAsed on November 17, 2010

Combined the following amounts of RNA with water to make 1ug RNA template in RT reactions


 * Sample ID || User ID || Date || Time || ng/ul || ul for 1ug || ul H2O ||
 * DNase FL1-3 || Default || 11/17/10 || 1:44 PM || 169.83 || 5.89 || 3.11 ||
 * DNase FL1-4 || Default || 11/17/10 || 1:44 PM || 166.36 || 6.01 || 2.99 ||
 * DNase FL1-5 || Default || 11/17/10 || 1:45 PM || 158.92 || 6.29 || 2.71 ||
 * DNase FL2-1 || Default || 11/17/10 || 1:46 PM || 177.33 || 5.64 || 3.36 ||
 * DNase FL2-2 || Default || 11/17/10 || 1:46 PM || 167.56 || 5.97 || 3.03 ||
 * DNase FL3-1 || Default || 11/17/10 || 1:47 PM || 132.58 || 7.54 || 1.46 ||
 * DNase FL3-2 || Default || 11/17/10 || 1:47 PM || 71.8 || 13.93 || 0.00 ||
 * DNase FL3-3 || Default || 11/17/10 || 1:48 PM || 94.64 || 10.57 || 0.00 ||

- Added 1ul OligoDT and Incubated at 70 degrees for 5min --ice immediately

Mastermix (10 samples) 5xM-MLV buffer: 50ul dNTP: 50ul M-MLV RT: 10ul Water: 40ul

DNase treatment of FL samples from November 5 & 8
 * November 17, 2010 **

Combined the following volumes of RNA and Water based on concentration so as not to exceed 10ug RNA/45ul


 * Sample ID || ng/ul || ul RNA || ul Water ||
 * FL1-3 || 221.32 || 45 || 0 ||
 * FL2-1 || 627.65 || 16 || 29 ||
 * FL2-2 || 343.51 || 29 || 16 ||
 * FL3-1 || 170.1 || 45 || 0 ||
 * FL3-2 || 92.31 || 45 || 0 ||
 * FL3-3 || 123.07 || 45 || 0 ||
 * FL1-4 || 449.77 || 22 || 23 ||
 * FL1-5 || 384.65 || 26 || 19 ||

Added 5ul of 10x DNase Turbo buffer and 1ul DNAse Turbo Incubate 37 degrees C for 30min Add 5.5ul Inactivation reagent Spin 10,000xg for 1.5min Transfer supt. to new tube and spec.

Results:


 * Sample ID || User ID || Date || Time || ng/ul || A260 || A280 || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || 340 raw ||
 * DNase FL1-3 || Default || 11/17/10 || 1:44 PM || 169.83 || 4.246 || 2.283 || 1.86 || 1.77 || 40 || 230 || 2.395 || 0.013 ||
 * DNase FL1-4 || Default || 11/17/10 || 1:44 PM || 166.36 || 4.159 || 2.153 || 1.93 || 1.78 || 40 || 230 || 2.338 || 0.028 ||
 * DNase FL1-5 || Default || 11/17/10 || 1:45 PM || 158.92 || 3.973 || 2.068 || 1.92 || 1.81 || 40 || 230 || 2.2 || 0.027 ||
 * DNase FL2-1 || Default || 11/17/10 || 1:46 PM || 177.33 || 4.433 || 2.341 || 1.89 || 1.85 || 40 || 230 || 2.394 || 0.031 ||
 * DNase FL2-2 || Default || 11/17/10 || 1:46 PM || 167.56 || 4.189 || 2.189 || 1.91 || 1.72 || 40 || 230 || 2.431 || 0.027 ||
 * DNase FL3-1 || Default || 11/17/10 || 1:47 PM || 132.58 || 3.314 || 1.71 || 1.94 || 1.35 || 40 || 230 || 2.447 || 0.176 ||
 * DNase FL3-2 || Default || 11/17/10 || 1:47 PM || 71.8 || 1.795 || 0.919 || 1.95 || 1.23 || 40 || 230 || 1.455 || 0.023 ||
 * DNase FL3-3 || Default || 11/17/10 || 1:48 PM || 94.64 || 2.366 || 1.247 || 1.9 || 1.26 || 40 || 230 || 1.881 || 0.088 ||

And here is the final installment of the hard clam/QPX RNA extractions using TriReagent...
 * November 9, 2010**


 * Sample ID || User ID || Date || Time || ng/ul || A260 || A280 || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || 340 raw ||
 * MA1-3 || Default || 11/9/10 || 11:48 AM || 335.15 || 8.379 || 4.548 || 1.84 || 2.46 || 40 || 230 || 3.408 || 0.002 ||
 * MA1-5 || Default || 11/9/10 || 11:49 AM || 176.71 || 4.418 || 2.608 || 1.69 || 2.57 || 40 || 230 || 1.722 || 0.014 ||
 * MA2-4 || Default || 11/9/10 || 11:49 AM || 452.18 || 11.304 || 6.036 || 1.87 || 2.13 || 40 || 230 || 5.31 || 0.043 ||
 * MA2-5 || Default || 11/9/10 || 11:50 AM || 379.45 || 9.486 || 5.077 || 1.87 || 2.4 || 40 || 230 || 3.952 || -0.057 ||
 * MA3-3 || Default || 11/9/10 || 11:50 AM || 213.62 || 5.341 || 2.96 || 1.8 || 2.48 || 40 || 230 || 2.154 || 0.027 ||
 * MA3-4 || Default || 11/9/10 || 11:50 AM || 246.55 || 6.164 || 3.514 || 1.75 || 2.52 || 40 || 230 || 2.449 || 0.011 ||
 * MA4-3 || Default || 11/9/10 || 11:51 AM || 230.18 || 5.754 || 3.172 || 1.81 || 2.51 || 40 || 230 || 2.29 || 0.013 ||
 * MA4-4 || Default || 11/9/10 || 11:51 AM || 163.11 || 4.078 || 2.344 || 1.74 || 2.62 || 40 || 230 || 1.554 || 0.012 ||
 * BX1-3 || Default || 11/9/10 || 11:52 AM || 403.66 || 10.092 || 5.618 || 1.8 || 2.3 || 40 || 230 || 4.395 || 0.017 ||
 * BX1-4 || Default || 11/9/10 || 11:53 AM || 260.08 || 6.502 || 3.516 || 1.85 || 2.39 || 40 || 230 || 2.725 || 0.021 ||
 * BX2-3 || Default || 11/9/10 || 11:53 AM || 240.71 || 6.018 || 3.428 || 1.76 || 2.43 || 40 || 230 || 2.477 || 0.016 ||
 * BX3-3 || Default || 11/9/10 || 11:54 AM || 266.55 || 6.664 || 3.73 || 1.79 || 2.38 || 40 || 230 || 2.803 || 0.012 ||

More hard clam/QPX RNA extractions using TriReagent:
 * November 8, 201**0


 * Sample ID || User ID || Date || Time || ng/ul || A260 || A280 || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || 340 raw ||
 * BX1-1 || Default || 11/8/10 || 2:20 PM || 91.4 || 2.285 || 1.463 || 1.56 || 2.22 || 40 || 230 || 1.027 || -0.007 ||
 * BX1-2 || Default || 11/8/10 || 2:21 PM || 65.54 || 1.639 || 0.978 || 1.67 || 1.82 || 40 || 230 || 0.9 || 0.023 ||
 * BX2-1 || Default || 11/8/10 || 2:21 PM || 94.22 || 2.355 || 1.406 || 1.68 || 1.89 || 40 || 230 || 1.247 || 0.022 ||
 * BX2-2 || Default || 11/8/10 || 2:22 PM || 137.09 || 3.427 || 1.957 || 1.75 || 1.98 || 40 || 230 || 1.733 || 0.022 ||
 * BX3-1 || Default || 11/8/10 || 2:22 PM || 46.78 || 1.17 || 0.714 || 1.64 || 1.72 || 40 || 230 || 0.678 || 0.065 ||
 * BX3-2 || Default || 11/8/10 || 2:23 PM || 279.37 || 6.984 || 4.041 || 1.73 || 2.22 || 40 || 230 || 3.142 || 0.02 ||
 * BX4-1 || Default || 11/8/10 || 2:24 PM || 329.88 || 8.247 || 4.541 || 1.82 || 1.53 || 40 || 230 || 5.373 || 0.672 ||
 * BX4-2 || Default || 11/8/10 || 2:24 PM || 193.62 || 4.841 || 2.73 || 1.77 || 1.57 || 40 || 230 || 3.083 || 0.388 ||
 * FL1-4 || Default || 11/8/10 || 2:25 PM || 449.77 || 11.244 || 6.223 || 1.81 || 2.18 || 40 || 230 || 5.157 || 0.049 ||
 * FL1-5 || Default || 11/8/10 || 2:25 PM || 384.65 || 9.616 || 5.318 || 1.81 || 2.25 || 40 || 230 || 4.275 || 0.046 ||

Some of the 260/280 ratios are still a bit low. I think this is because, again, there was not a lot of gill tissues left in the tube from before and in order to get a good quality extraction I need to have a bit more. I think I will extract more samples from BX site 1 and one more from BX site 3. I will also extract new samples for MA, but this time I will extract NEW samples and not the second half of the remaining tissue from the previous extraction.


 * November 5, 2010**

FL hard clam gill RNA extractions take 2: Overview: So the reason for this do-over is that the RNA extractions from October 29, 2010 did not yield the quality of RNA I need to proceed with qPCR. The reasoning in my mind was that it was either my fault for not doing the extractions as continuously as I should have, or the sample quality is low and the RNA is already degraded. To find out the answer, I will be extracting a subset of the samples from that round extractions again. FL was chosen because they are most likely to have an immune response (judging from previous data) from which I can then test my SPI primers.

RNA-later RNA extraction protocol was carried out to a tee!


 * Sample ID || User ID || Date || Time || ng/ul || A260 || A280 || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || 340 raw ||
 * FL1-1 || Default || 11/5/10 || 2:09 PM || 78.02 || 1.95 || 1.098 || 1.78 || 0.37 || 40 || 230 || 5.211 || 0.006 ||
 * FL1-2 || Default || 11/5/10 || 2:09 PM || 55.51 || 1.388 || 0.792 || 1.75 || 1.46 || 40 || 230 || 0.949 || -0.002 ||
 * FL1-3 || Default || 11/5/10 || 2:10 PM || 221.32 || 5.533 || 3.022 || 1.83 || 2.12 || 40 || 230 || 2.611 || 0.01 ||
 * FL2-1 || Default || 11/5/10 || 2:10 PM || 627.65 || 15.691 || 8.167 || 1.92 || 2.06 || 40 || 230 || 7.634 || 0.158 ||
 * FL2-2 || Default || 11/5/10 || 2:11 PM || 343.51 || 8.588 || 4.513 || 1.9 || 1.97 || 40 || 230 || 4.37 || 0.048 ||
 * FL3-1 || Default || 11/5/10 || 2:11 PM || 170.1 || 4.253 || 2.189 || 1.94 || 1.45 || 40 || 230 || 2.942 || 0.25 ||
 * FL3-2 || Default || 11/5/10 || 2:12 PM || 92.31 || 2.308 || 1.243 || 1.86 || 1.39 || 40 || 230 || 1.659 || 0.019 ||
 * FL3-3 || Default || 11/5/10 || 2:12 PM || 123.07 || 3.077 || 1.609 || 1.91 || 1.52 || 40 || 230 || 2.024 || 0.17 ||

RNA looks much much MUCH better! However, I think that both of my ideas for why the last round was so bad are both right/wrong. From these results, samples 1-1 and 1-2 are both moderate in that they have very low concentrations (they were also resuspended in 30ul of water instead of 75ul like the others) and their ratios are lower than other samples. These samples also had the smallest amount of tissue left in the tube!

Conclusion: 1. I should have extracted the other samples in smaller batches and to completion instead of breaking for classes, teaching, and other extractions. 2. I was not accustomed to working with gill tissue and it looks like you need more tissue than I thought and small amounts of tissue dont provide a robust amount of RNA. 3. I think I will pick two more FL samples from site one to replace 1-1 and 1-2.

Note to self: the following 3 entries are place holders with bullet points of what happened and need to be more completely written up...

I need to generate some cDNA from the hard clam samples so I can test my primers during Physiology lab tomorrow.
 * November 1, 2010 **

Promega Turbo DNA-free protocol: 1. Combine: 25ul RNA, 25ul sterile water, 5ul 10x Turbo DNase buffer, 1ul Turbo DNase 2. Incubate 37 degrees C 30min 3. Add 1ul Turbo DNase (rigorous protocol) 4. Incubate 37 degrees C 30min 5. Add 5.5ul DNase inactivation reagent. 6. Flick with finger to mix and incubate at RT for 2min 7. Spin 10,000g for 1.5min. 8. Move supt. to new tube. 9. Spec 2ul on nanodrop


 * Sample ID || User ID || Date || Time || ng/ul || A260 || A280 || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || 340 raw ||
 * Max 4 DNase || Default || 11/1/10 || 1:00 PM || 172.32 || 4.308 || 2.244 || 1.92 || 1.65 || 40 || 230 || 2.618 || 0.02 ||
 * Max 5 DNase || Default || 11/1/10 || 1:00 PM || 73.09 || 1.827 || 0.997 || 1.83 || 1.22 || 40 || 230 || 1.499 || -0.002 ||
 * Max 6 DNase || Default || 11/1/10 || 1:01 PM || 44.17 || 1.104 || 0.618 || 1.79 || 1.02 || 40 || 230 || 1.087 || -0.026 ||
 * Max 7 DNase || Default || 11/1/10 || 1:01 PM || 47.22 || 1.181 || 0.673 || 1.75 || 0.79 || 40 || 230 || 1.489 || 0.065 ||
 * Max 8 DNase || Default || 11/1/10 || 1:02 PM || 45.37 || 1.134 || 0.624 || 1.82 || 1.08 || 40 || 230 || 1.048 || -0.012 ||
 * Max 9 DNase || Default || 11/1/10 || 1:02 PM || 111.74 || 2.793 || 1.462 || 1.91 || 1.54 || 40 || 230 || 1.81 || 0.011 ||
 * Max 10 DNase || Default || 11/1/10 || 1:03 PM || 73.46 || 1.837 || 0.977 || 1.88 || 1.28 || 40 || 230 || 1.437 || 0.056 ||

RT Rxn. (MAX 4 only) Note: only using one sample so I will have cDNA to use in class tomorrow. I will do a larger RT reaction later on all of the samples at the same time to control for master mix biases. 1. 5uL of DNase RNA (~1ug) was combined with 1uL oligo dT and 4uL nuclease free water in a 0.5ml PCR tube. 2. Sample was spun to collect contents at the bottom of the tube and then incubated at 70 degrees Celsius for 5min. 3. Sample was spun down again and kept on ice. 3. 5uL of M-MLV 5x reaction buffer, 5uL dNTPs, 1uL M-MLV RT (the reverse transcriptase), and 4uL of nuclease free water was added to the sample. 4. Sample was spun again and then placed in the thermocycler (42 degreesC for 60min followed by 70 degreesC for 3min). 5. Samples were spun down and stored on ice.

Spun down and resuspended RNA extractions from _DATE
 * October 29, 2010 **

Results: Not good


 * Sample ID || User ID || Date || Time || ng/ul || A260 || A280 || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || 340 raw ||
 * FL1-1 || Default || 10/29/10 || 10:39 AM || 76.81 || 1.92 || 1.132 || 1.7 || 1.34 || 40 || 230 || 1.434 || 0.033 ||
 * FL1-2 || Default || 10/29/10 || 10:40 AM || 31.61 || 0.79 || 0.48 || 1.65 || 1.17 || 40 || 230 || 0.674 || 0.014 ||
 * FL1-3 || Default || 10/29/10 || 10:40 AM || 27.08 || 0.677 || 0.413 || 1.64 || 1.11 || 40 || 230 || 0.612 || 0.01 ||
 * FL12-1 || Default || 10/29/10 || 10:41 AM || 51.74 || 1.294 || 0.775 || 1.67 || 1.42 || 40 || 230 || 0.911 || 0.05 ||
 * FL12-2 || Default || 10/29/10 || 10:42 AM || 105.67 || 2.642 || 1.55 || 1.7 || 1.42 || 40 || 230 || 1.865 || 0.049 ||
 * FL13-1 || Default || 10/29/10 || 10:43 AM || 237.51 || 5.938 || 3.27 || 1.82 || 1.33 || 40 || 230 || 4.47 || 0.488 ||
 * FL3-2 || Default || 10/29/10 || 10:44 AM || 53.29 || 1.332 || 0.771 || 1.73 || 0.94 || 40 || 230 || 1.421 || 0.252 ||
 * FL3-3 || Default || 10/29/10 || 10:44 AM || 34.66 || 0.867 || 0.525 || 1.65 || 0.69 || 40 || 230 || 1.261 || 0.179 ||
 * MA1-1 || Default || 10/29/10 || 10:45 AM || 174.19 || 4.355 || 2.482 || 1.75 || 2.02 || 40 || 230 || 2.154 || 0.035 ||
 * MA1-2 || Default || 10/29/10 || 10:45 AM || 25.15 || 0.629 || 0.389 || 1.61 || 1.15 || 40 || 230 || 0.545 || 0.025 ||
 * MA2-1 || Default || 10/29/10 || 10:46 AM || 18.45 || 0.461 || 0.272 || 1.69 || 0.8 || 40 || 230 || 0.575 || 0.019 ||
 * MA2-2 || Default || 10/29/10 || 10:46 AM || 7.99 || 0.2 || 0.107 || 1.87 || 0.52 || 40 || 230 || 0.385 || 0.006 ||
 * MA3-1 || Default || 10/29/10 || 10:47 AM || 385.04 || 9.626 || 5.358 || 1.8 || 2.11 || 40 || 230 || 4.555 || 0.082 ||
 * MA3-2 || Default || 10/29/10 || 10:47 AM || 399.03 || 9.976 || 5.589 || 1.79 || 2.08 || 40 || 230 || 4.793 || 0.089 ||
 * MA4-1 || Default || 10/29/10 || 10:48 AM || 22.27 || 0.557 || 0.333 || 1.67 || 1.07 || 40 || 230 || 0.52 || 0.014 ||
 * MA4-2 || Default || 10/29/10 || 10:48 AM || 12.95 || 0.324 || 0.193 || 1.67 || 0.84 || 40 || 230 || 0.387 || -0.004 ||
 * BX1-1 || Default || 10/29/10 || 10:49 AM || 33.71 || 0.843 || 0.511 || 1.65 || 1.23 || 40 || 230 || 0.687 || 0.017 ||
 * BX1-2 || Default || 10/29/10 || 10:49 AM || 25.28 || 0.632 || 0.375 || 1.69 || 1.15 || 40 || 230 || 0.551 || 0.004 ||
 * BX2-1 || Default || 10/29/10 || 10:50 AM || 68.16 || 1.704 || 0.963 || 1.77 || 1.47 || 40 || 230 || 1.156 || 0.036 ||
 * BX2-2 || Default || 10/29/10 || 10:50 AM || 78.96 || 1.974 || 1.192 || 1.66 || 1.62 || 40 || 230 || 1.219 || 0.152 ||
 * BX3-1 || Default || 10/29/10 || 10:51 AM || 16.89 || 0.422 || 0.24 || 1.76 || 0.92 || 40 || 230 || 0.46 || 0.016 ||
 * BX3-2 || Default || 10/29/10 || 10:51 AM || 167.42 || 4.186 || 2.721 || 1.54 || 1.35 || 40 || 230 || 3.109 || 0.544 ||
 * BX4-1 || Default || 10/29/10 || 10:52 AM || 431.15 || 10.779 || 5.966 || 1.81 || 1.6 || 40 || 230 || 6.727 || 0.648 ||
 * BX4-2 || Default || 10/29/10 || 10:52 AM || 199.28 || 4.982 || 2.807 || 1.77 || 1.67 || 40 || 230 || 2.977 || 0.264 ||

For whatever reason, the RNA quality of these samples is very poor. Yield is low and 260/280 ratios are nowhere near the 1.8-2.0 range that I feel comfortable using as clean non-degraded RNA. Fortunately, I still have the second half of the tissue that I saved in RNA-later. The plan is to try extracting one sample set at a time (ie. FL only). One of my worries is that I was trying to extract too many samples at a time with too many other obligations and the protocol was not carried out as fluidly as necessary. The other option is that these samples were received in May (i think) and they were kept in the fridge until I extracted them so the RNA might not be properly preserved and/or they were not properly preserved to begin with.

Purification of hard clam hemocytes for RNA isolation
 * October 27, 2010 **

The purpose of this experiment is to isolate a large enough population of hemocytes for RNA extraction to make libraries. -bled 6 individuals from each site (2-3ml total blood). -immediately added 3ml seawater w/ pen/strep -incubated o/n at 12 degrees -washed 3x seawater w/ pen/strep -added 3ml seawater w/ pen/strep and looked at the results:

Fig1. MA4 blood plated on polylysine plates at the 17hours. 20X magnification. Fig2. MA4 hemocytes plated on polylysine plates at 17hours. 40x magnification.

Fig2. BFX1 blood plated on polylysine plates at 17 hours. 20X magnification

October 26, 2010 QPX M. mercenaria Gill tissue challenge

This is a Preliminary experiment to dissect gill tissue and then challenge it with QPX in a test tube and see if we can get any meaningful transcript data out of ti. We are using three different strains of QPX, S-1, ATTC, and TD8-81, plus a control using just seawater w/o QPX.

Gill tissue from clam BFX 9 was used in all treatments.

Three treatments for each strain 1. QPX in seawater 2. Gill tissue exposed to QPX in seawater. 3. QPX in the presence of gill tissue (After challlenge is complete, Isolate QPX from the supernatent of treatment 2.)

Incubate 24hrs at 37 degrees Celsius.

Spin treatments 1 and 3 at 14k for 10 min to pellet QPX

Add 1ml of TriReagent to treatments 1 and 3, and 5ooml TriReagent to treatment 2 (easier to homogenize later). Store at -80.


 * October 15,****2010 **

More TriReagent RNA extractions of M. mercenaria gills from QPX samples (AUGUST/SEPTEMBER...) Also measured of concentration for RNA samples purified on September 24, 2010 Note: These samples were stored as pellets in 75% ETOH at -80degrees. I spun the pellets down for 5min 12K and resuspended in 50uL DEPC treated water.


 * October 8, 2010**

RNA extractions from pinto abalone larvae. Contiuned from OA treatment October 1, 2010

-For this extraction I am just going to see what kind of yield (both amount and quality) or RNA I can get from pinto abalone larvae. Since I've never done this before and have no idea how much RNA yield to expect, I will be extracting RNA from three different amounts of larvae.

Sample 1: 380pCO2 A Sample 2: pooled 380pCO2 B&C Sample 3: pooled 840pCO2 A,B,&C

All samples were taken form september 28, 2010. An individual sample should contain ~50 larvae, so the RNA extraction samples should contain 50, 100, and 150 larvae respectively.

Followed protocol for RNA extraction using TriReagent Note: homogenized in 200uL before adding additional 800ul of trireagent. Note: Larvae were stored in RNAlater and float to the top. I tried spinning for 10s at 14k and the larvae only moved partially down the solution. In the future I recommend NOT spinning and leaving the larvae collected at the surface while pipetting the RNAlater out from the bottom.


 * Results! ||  ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||   ||
 * Sample ID || User ID || Date || Time || ng/ul || A260 || A280 || 260/280 || 260/230 || Constant || Cursor Pos. || Cursor abs. || 340 raw ||
 * AbLarv 1 || Default || 10/8/10 || 1:18 PM || 8.27 || 0.207 || 0.129 || 1.6 || 0.16 || 40 || 230 || 1.297 || 0.012 ||
 * AbLarv 2 || Default || 10/8/10 || 1:19 PM || 21 || 0.525 || 0.348 || 1.51 || 0.14 || 40 || 230 || 3.777 || 0.009 ||
 * AbLarv 3 || Default || 10/8/10 || 1:20 PM || 59.71 || 1.493 || 0.922 || 1.62 || 0.5 || 40 || 230 || 2.991 || 0.004 ||
 * AbLarv 1 || Default || 10/8/10 || 1:18 PM || 8.27 || 0.207 || 0.129 || 1.6 || 0.16 || 40 || 230 || 1.297 || 0.012 ||
 * AbLarv 2 || Default || 10/8/10 || 1:19 PM || 21 || 0.525 || 0.348 || 1.51 || 0.14 || 40 || 230 || 3.777 || 0.009 ||
 * AbLarv 3 || Default || 10/8/10 || 1:20 PM || 59.71 || 1.493 || 0.922 || 1.62 || 0.5 || 40 || 230 || 2.991 || 0.004 ||

-I definitely got RNA, however it is not as clean as I would have hoped. My thought is that I was being a little bit too greedy when I was removing the upper phase after adding chloroform and may have sucked up some debris. I was not expecting this much RNA and wanted every last drop of that upper phase. I'm hoping that by doing this again and not coming as close to the interphase will help clean it up. I can also perform an additional EtOH wash to make sure.

-RNA concentrations are not as linear as expected considering the extractions should represent 50,100, and 150 larvae....in particular the "150" larvae sample is pretty high. This might be because these larvae came from a different treatment group or the jar contained a higher concentration of larvae. I have asked Liza for her larval counts from the samples that we took in parallel to these to get a better estimation of larval quantity.


 * October 5, 2010**

Hard Clam QPX continued...

We will be doing RNA extractions during the Envirophys lab today, so instead of using some random tissue, I will be using some of the gill tissue from the QPX study....why not right?

Today I just prepared the samples by cutting the gill tissue in half as before, and storing it at -80 until next weeks lab to do the RNA extractions.


 * October 4, 2010**

__Abalone Antibiotic Challenge:__ Note: This experiment is along the same lines as the differential centrifugation experiment with Percoll gradients in that we are trying to selectively isolate pure strains of rickettsia. In this case, we are going to try and utilize the differential antibiotic resistance properties of the three strains.

__Approach:__ 4 treatments x 4reps per treatment. Treatment 1: inject w/ saline solution (2% NaCl) Treatment 2: inject w/ clarithromycin (previous resistance shown for "new" strain) Treatment 3: inject w/ Rifampacin Treatment 4: inject w/ Erythromycin

__Animals:__ ~100 juvenal red abalone were "incubated" for several months (need to refer back to wetlab notebook for exact time) with 5 adult red abolone known to be infected with all three strains of rickettsia.

- 8 individuals were selected on (refer to histo notes for date) for histo sampling (head, foot, DG, PE). Body weight, shell weight, and shell length were also taken. - All 8 individuals were positive for the new strain of rickettsia which is what we're most interested in, however, prevalence of the other two strains was varied. - Refer to Histo notes **10:24-(1-8)**

Antibiotic preparation: Note: Stock concentrations were calculated based on the assumption that we will be injecting abalone with 50ul of solution. Antibiotics are not soluble in water, so a 10x solubilization solution is required. 1:10 dilutions were made by diluting 1ml 10x solution in 9ml 2%saline solutions. 7 aliquots were made (one for each day of the week) and frozen at -20.


 * Antibiotic || Dose || Stock || 10x || Solubilize ||
 * Clarithromycin || 20mg/kg || 5.2mg/ml || 52mg/ml || EtOH ||
 * Rifampacin || 2mg/kg || 0.52mg/ml || 5.2mg/ml || MeOH ||
 * Erythromycin || 15mg/kg || 3.9mg/ml || 39mg/ml || MeOH ||

- Concentration of stock solutions were calculated from average body weight taken from the 8 pre-sampled individuals:

Avg BW = 13.02
 * # || TW || SW || BW ||
 * 1 || 15.1 || 4.6 || 10.5 ||
 * 2 || 14.3 || 4.1 || 10.2 ||
 * 3 || 16.65 || 4.09 || 12.56 ||
 * 4 || 20.67 || 5.18 || 15.49 ||
 * 5 || 13.7 || 6.5 || 7.2 ||
 * 6 || 20.3 || 5.2 || 15.1 ||
 * 7 || 15.89 || 3.8 || 12.09 ||
 * 8 || 28.99 || 8.0 || 20.99 ||

Injections: 50ul injections will be preformed daily for 14 days. This entry is a little bitter sweet because I was hoping to begin injections today but I cannot find clarithromycin. I know we had some when I last looked for it, but since then the fridge has been cleaned so I am assuming that it is sitting somewhere, very well organized, in a place I would never find it.

--ordered 100mg of clarithromycin


 * October 1, 2010 **

__OA-Pinto abalone__

The fine people at our Mukilteo station succeeded at spawning some pinto abalone and we were able to get a fairly generous number of larvae.

Approach x2:

1. Two OA treatments. 380ppm and 840ppm, 6 reps for each system. 2. Two care protocols for each treatment. Water changes everyday, water changes every other day. 3 reps for each system

-Abalone were spawned on 9/23/10 and were put in the OA systems on 9/27/10. Systems started out on a low flow rate to acclimate the larvae to new water and OA conditions. - First day of sampling began on 9/28/10 and was terminated on 10/1/10

Treatment Breakdown: 380A-C: 380pCO2, H2O Change everyday 380D-F: 380pCO2, H2O Changed everyother day 840A-C: 840pCO2, H2O Change everyday 840D-F: 840pCO2, H2O Change everyday

What are we sampling? 50ml/jar for live/dead analysis 50ml/jar for RNA extraction (concentration of larvae in the jars is ~1larvae/ml...so in theory, each tube should have 50 larvae)

Sampling Technique: -Dunk filter up and down to dislodge any larvae that might be stuck to the 100micron screen. -Use "plunger" to form homogenous mixture of larvae in the jar. -Submerge 50ml falcon tube to collect sample. -Samples were kept in cooler with icepacks for transport back to SAFS labs (samples were not "kept on ice" per se, just in a cool environment)

RNA preservation: -On Days 1&2 of sampling, I had collected larvae in 50ml falcon tubes, brought them back to SAFS, spun them down for 1min, 1000rpm in a swing bucket rotor, sucked off the water, added RNAlater, and transferred to screw cap tube.

-Days 3&4 of sampling, I prefilled screw cap tubes with 1ml RNAlater and took these with me to NMFS. I still took samples using 50ml falcon tubes, except this time I immediately filtered the larvae through a submerged 100 micron filter and pipetted them out directly into the RNA later. I have yet to test the RNA from the two sampling methods but this one is much MUCH easier and I think that the sooner you can get the larvae into RNAlater the better.

-All RNAlater samples are currently being stored at 4 degrees. I hope to do some extractions within the next week, but if not they will be moved to -80degrees.

Sampling results: -Larvae were beginning to settle by day 3 of sampling thus resulting in lower and lower concentrations of swimming larvae in our samples. -On the last day of sampling we sampled as usual, but left the jars unhooked from the system for ~3hrs to allow any setting or dead larvae to sink to the bottom. This, hopefully, gave us a pure sample of swimming larvae higher up in the water column. Larvae were then siphoned and pooled from all 6 jars/treatment onto a 100 micron screen and preserved in RNA later. Once my RNA extraction protocol is worked out, this sample should hopefully serve as an RNA pool from a very late time point that can be used to generate a cDNA library.


 * September 24, 2010 **

__Hard Clam QPX__

Note: Sam has already generated some preliminary data on this project. -Samples 1-3 from CA, MA, and MAX have already been extracted.

- I am going to continue w/ extractions and do a total of 10 samples of gill tissue from each site. - To get started, I am only going to process a small number of samples since I will also be figuring out where everything is in the lab etc... - I'll start by extracting RNA from gill tissue samples CA 4-9

RNA extraction using TriReagent: 1. Cut gill tissue in 1/2. Used one half for RNA extraction and placed the other half back at -80 degrees as a backup. 2. Add 300uL TriReagent 3. Homogenize with sterile blue pestle. 5. Incubate at RT for 5min 4. cf for 15', 12,000xg @ 4 degrees. 5. Transfer aqueus phase to new tube 6. Precipitate by adding 500ul Isopropanol 7. Incubate at RT 5min 8. Cf 12,000xg for 8min @ 4 degrees 9. Remove isopropanol and wash with 75% EtOH 10. Spin 5min 12,000xg @ 4 degrees 11. Remove EtOH and add 1ml fresh EtOH Note: I saw a nice big white pellet so I am going to stop here and store the RNA as a pellet until the remaining samples have caught up to this point before going ahead and solubilizing the RNA for cDNA synthesis. 12. Store at -80 in EtOH as a pellet.


 * September 2, 2010**

Exp: Rickettsia isolation by differential centrifugation:

-I am going to try using a Percoll solution to separate Rickettsia strains based on cell size. -Why use Percoll? From articles I found using similar methods, Percoll seemed to be the easiest to use in that it forms a self forming gradient upon centrifugation and is also reported to maintain cell viability. This is important if the cell isolation is successful because we would then be able to inject healthy abalone with pure strains of live Rickettsia w/o having to culture the pathogen.

Trial 1:
 * Made homogenization buffer: 0.33M Tris-Cl, 0.25M Sucrose

1. Dissect post esophagus from a single infected red abalone. 2. Homogenize in 3ml homogenization buffer w/ a Dounce homogenizer 3. Spin homogenate at 210xg (~1000rpm) for 10' to pellet cell debris 4. Remove supt. and layer it onto 27ml of 30% Percoll 30% Percoll = 9ml Percoll solution, 1ml 1.5M NaCl, 17ml H2O 5. Spin 25,000xg for 1hr 6. Take 1ml fractions and stain w/ hemastain kit. Look

Results: 1. Differential centrifugation resulted in 4 distinct visual layers. ~5ml upper layer of clear liquid ~5ml middle layer opaque liquid ~1ml disctinct debris layer that was located ~10ml down the gradient...presumably the leading edge of the homogenate ~ remaining clear liquid 2. Cells only appeared in the 1ml "debris" layer 3. Difficult to distinguish rickettsia from everything else.

Future Exp: Repeat as before but spin Percoll gradient for 2hrs. Isolate "debris" layer, and spin again for 2-3hrs in a 40% percoll gradient.