Emma's+Notebook

include component="page" wikiName="genefish" page="Emma's winter notebook 2014" editable="1" wrap="1" include component="page" wikiName="genefish" page="Emma's Brest Lab Notebook" editable="1" wrap="1" include component="page" wikiName="genefish" page="Emma's Summer Notebook 2013" editable="1" wrap="1" include component="page" wikiName="genefish" page="Emma's Spring Notebook 2013" editable="1" wrap="1" include component="page" wikiName="genefish" page="Emma's Lab Notebook Winter 2013" editable="1" wrap="1" include component="page" wikiName="genefish" page="Emma's Lab Notebook Autumn 2012" editable="1" include component="page" wikiName="genefish" page="Emma's notebook Spring 2012" editable="1" wrap="1" include component="page" wikiName="genefish" page="Emma's Lab Notebook Winter 2012" editable="1"

include component="page" wikiName="genefish" page="Emma's Lab Notebook Autumn 2011" title="Emma's Lab Notebook Autumn 2011" editable="1"

include component="page" page="Emma's Lab Notebook Spring 2011" editable="1" include component="page" page="Emma's Lab Notebook Winter 2011" editable="1"

include component="page" page="Emma's Lab Notebook Autumn 2010" editable="1"

**September 8, 2010** __OA at NWFSC__ Experimental plan with Shallin and Elene. Day 0 = Monday, September 13. pCO2 treatments (6 replicates): 280, 380, 740 ppm. 3 replicates at 2000 ppm just for development data. Day 0, 9/13: fertilization; check development at 0.5, 2, and 5 hours. Data: demographics, RNA. Systems are static. Dat 1, 9/14: Switch to flow-through. Data: Demographics, RNA Day 2, 9/15: terminate 2000 ppm at 48 hours. Clean jars. Data: demographics, DNA. Day 4, 9/17: Clean jars. Data: demographics, DNA, RNA. Day 7, 9/20: Clean jars. Data: demographics, DNA, RNA. Day 10, 9/23: Clean jars. Data: demographics, DNA, RNA. Day 14, 9/27: Place shell fragments for settlement in bottom of jars. Clean jars. Data: demographics, DNA, RNA. Day 17, 9/30: Clean jars. Data: demographics, DNA, RNA. Day 21, 10/4: Clean jars. Data: demographics, DNA, RNA.

__Sperm motility at NWFSC__ Plan for preliminary sperm motility experiment with A. Bruner and A. Dittman. Will check cell densities of C. gigas sperm on 9/9 and make standard curve. Video capture 9/10.

__OADev__ Repeat of qPCR 9/3 on Friedman lab qPCR thermalcycler. See 9/3 for details.

**September 7, 2010** __OADev__ qPCR from 9.3.10 had funky fluorescence. Repeat PCR (same sheet as 9.3). media type="custom" key="6854805"

Still weird fluorescence. qPCR to check SYTO 13 (50 uM) made 8.31.10. Made new SYTO stock - 1 uL 5mM SYTO 13 in 99 uL DMSO. qPCR in replicate with master mixes made from 2 different SYTO dilutions. media type="custom" key="6858065" fluorescence is a problem with the qPCR machine.

__OA at NWFSC__ 1 pm: dropped off oysters collected at Big Beef Creek on 9.6. Oysters are at ~20C in flowing water.

**September 6, 2010** __Prep for spawning__ Collected ~23 adult C. gigas from Big Beef Creek. Tide was high (~1:30 pm). Opened a few oysters to check ripeness before collecting. Oysters on river side of bridge above tide were barely ripe. More ripeness in subtidal oysters under bridge and along the beach in the bay. Oysters were collected from these 2 locations and transported back to UW on ice. Once back at SAFS, put oysters in seawater tank with air and a pump to create water flow.

**September 3, 2010** __OADev__ Exported qPCR data from 9/1 and 9/2 with manual threshold of 0.011. Too much variation to tell if differences are significant or not. Samples that need to be rerun (due to low efficiency or failed amplification): EF1: 840 C2 CytP450: 380A2, 380 A1, 840 C1

qPCR of rerun samples and all samples for duplicate. Also included Hsp70 (CM primers). media type="custom" key="6834015"

__T/Vt__ Samples inoculated with Vt at 25C: A,G,D,H Samples inoculated with Vt at 12C: J,K,C,L Controls (unclear which are at which T): B,E,F,I,M,N,O,P - but we think the 25C controls are F,E,B,I and 12C controls are O,M,N,P. Exported qPCR data from 9/1 & 9/2 with manual threshold of 0.019. Samples that need to be rerun are: EF1: A,D,H,P cjunk: A,D,P,F,G,I NfkB: all

**September 2, 2010** __OADev__ ** qPCR of 380 A1, A2, B1 and 840 A1, B1, C1, C2 (cDNA). Genes = BPI, CytP450, Prx6, and Hsp70. ** media type="custom" key="6823999" Results: BPI did not amplify - need to check paper for published annealing temp, etc.; CytP450 & Prx6 were fine; Hsp70 had a terrible melt curve. SW says that the primer set is bad in general and gave me newly designed primers (from Christina Miller). __ ** T/Vt ** __ ** Reverse transcription (see 11/6/09) of 7.16 J & K. ** ** qPCR of T/Vt 7.16 cDNA with c-jun kinase and NfKB (also EF1a for J & K). Tested new Hsps70 primers (from CM) with some unknown cDNA. ** media type="custom" key="6825755"

**September 1, 2010** __OADev & T/Vt__ ** DNase (standard protocol) of 380 C1 and 7.16 J & K. ** ** Reverse transcription (MMLV see 11/6/09) of clean DNased samples from 8/30 and 8/31 (n=21). Samples in welled plated in following order: **
 * 7.16 L || 840 C1 || 7.16 G ||
 * 7.16 B || 7.16 N || 840 B1 ||
 * 7.16 F || 840 C2 || 7.16 C ||
 * 7.16 P || 840 A1 || 380 B1 ||
 * 7.16 H || 7.16 M || 7.16 I ||
 * 7.16 A || 380 A1 ||  ||
 * 7.16 E || 7.16 O ||  ||
 * 7.16 D || 380 A2 ||  ||

Diluted all cDNA in 225 uL water. Transferred to 1.5 mL tubes & stored at -20.

qPCR of cDNA (gene = EF1a) and of DNased RNA (diluted 1:20) 380C1 and 7.16 J & K (gene = 18s). media type="custom" key="6823797" Still contamination in 380C1, but J & K are clean.

Made sample database of all RNA, cDNA and samples.

**August 31, 2010** __OADev & T/Vt__ ** qPCR results from 8.30: Some samples were clean and some had gDNA contamination. Clean samples: 380 A1, 380 A2, 840 C2, 7.16 A, D, H, O and P. All negatives were clean and gDNA controls amplified more than most of the RNA samples. Chose samples with the highest gDNA levels, diluted them 1:20 in nanopure water and did qPCR with 18s primers. Samples picked for re-amp. were: 380 C1, 7.16 B, F, I, and N. ** media type="custom" key="6806273"

Still evidence of contamination in all samples tested at 1:20 dilution. Re-DNase (standard protocol, 1 uL DNase, 5 uL buffer, incubate 37C for 30 min) all samples that showed evidence of gDNA contamination in qPCR from 8.30. qPCR of re-DNased samples with 18s primers. media type="custom" key="6816203" All samples clean except for 380 C1. Will re-DNase 380C1 and 7.16 J &K, which were missed yesterday.

**August 30, 2010** __OADev & T/Vt__ ** Extracted RNA from OADev samples using Tri Reagent. Extracted 4 from each CO2 treatment. For 380 ppm: A1, B1, C1, A2. For 840 ppm: A1, B1, C1, C2. 380 ppm C1 spilled ~1/2 volume during vortexing after chloroform addition. **

Found RNA concentrations using Nanodrop for extracted OAdev sample & 16 RNA samples (extracted at FHL) from T/Vt trial 7.16. Did concentrations of 3 aliquots of 1.5 uL from each sample.

media type="custom" key="6800423" 3 concentrations were averaged unless one was anomalous, then is was discarded from calculations. From averaged concentration, determined how many uL would be needed for 10 ug. If >50 uL, then used entire sample for DNasing (all RNA samples are 50 uL); if <50 uL, then made up difference with DEPC-treated H2O. Dnased samples using Ambion's rigorous protocol (details March 18, 2010).

qPCR of all RNA samples with 18s primers to test for gDNA contamination (4 negative controls, 2 positive controls of C. gigas DNA). media type="custom" key="6806211"

**August 27-30, 2010** __LD50: C. gigas larvae + V. tubiashii__ ** Counted larvae in the am to determine live vs dead. Same protocol as 8.26. Did 4x on 8.26-28, switched to 10x on 8.29 & 8.30. Data here. ** ** In a number of the samples, fewer larvae were swimming as of 8.27 (see data for details). Ciliates first observed on 8.30 in chambers C, N, and B. Larvae were not counted in K & D on 8.30 because they had all been dead since 8.27. **

**August 26, 2010** __LD50: C. gigas larvae + V. tubiashii__ ** Got ~1x10^6 Cg larvae from Taylor yesterday (8/25). larvae were spawned on Monday (8/23). Divided larvae equally between 10 treatment chambers (1.5 L). Chambers were filled with seawater and aerated. ** ** 10 am today inoculated 8 treatment chambers with Vt. Inoculation at 4 concentrations in duplicate: 10^6, 10^5, 10^4, and 10^3 CFU/mL. 2 chambers left as controls. Before inoculation, homogenized larvae in water column with "plunger" and sampled 10 mL. Took 3 aliquots from those 10 mL and put in 3 wells of a depression slide. Counted larvae in 3 fields of view at 4x. Determined live vs. dead (xls "LD 50 mortality data"). Larvae appeared healthy and most were swimming actively. They had bivalved shells and velums. **

**July 27, 2010** **__IMPORTANT NOTE__** ** Until this week Lillian was not cleaning water collection jars used to sample microbe. communities outside the hatchery. **

**July 13, 2010** __Cg Larvae - Vt & T trial 2__
 * 8 am **
 * Set one tote to 12C and the other to 25 C using chiller & heater, respectively. Later in the day Elene inoculated 4 chambers in each tote. **
 * 9 pm **
 * Fed larvae. Divided ~ 125 mL T.iso/Plya between 16 chambers. **

** __Cg Larvae - Vt & T trial 2__ ** ** New larvae from Taylor. 9-day old, ~ 2 million. Divided between 16 larval chambers in 2 separate totes. **
 * July 12, 2010**

__Cg Larvae - Vt trial 1__ 8:25 am neutral red staining of larvae from each chamber 50 Vt colonies at 10^-3 4 pm ended experiment. Sampled all remaining larvae from remaining chambers. T=20C salinity = 31 ppt pH <8
 * July 9, 2010**


 * July 8, 2010**
 * __Cg Larvae - Vt trial 1__ **
 * 7: 40 am **
 * started 6 hour Vt incubation. Inoculated new media ( 25 mL of 0.25% tryptone and seawater) with 100 uL of 24 hour Vt culture from 7/7. Placed culture on rocker in incubator (26C). **
 * Counted colonies from yesterday's Vt used for inoculating larvae. Counted colonies on both 10^-3 concentration plates - n=109 & 74. According to equation CFU/mL = # colonies * 1/0.1 * dilution this 183 * 10^6 CFU/mL. **


 * 8:10 am **
 * Started neutral red incubation of 50 mL larvae + 50 uL 1% neutral red. **


 * 11 am **
 * Emptied, rinsed and replaced banjo filters in all chambers. Sampled larvae. There were very few larvae left in H so sampled all and now only have 7 chambers for remaining part of trial. **
 * T: 23C **
 * salinity: 30 ppt **
 * pH: ~8 **


 * 1:40 pm **
 * boiled 4 mL of Vt for 1 hour to heat kill. Made 5 serial dilutions: 1 mL Vt in 9 mL sterile water. Spread 2 plates each for last 3 dilutions - 10^-3, -4, and -5. Put in 25C incubator overnight. **
 * 2:10 pm **
 * Neutral red microscopy: photographed and measured larvae from all 8 chambers. **
 * 4 pm **
 * Inoculated treatment chambers (A, B, D & E) and put algae (T. iso + Plya, 50 mL between 7 containers) in all chambers. **

Ciliates observed in larvae from E.
 * July 7, 2010**
 * __Cg Larvae - Vt trial 1__ **
 * Temp: 21C **
 * salinity: 31 ppt **
 * pH: ~8 **
 * Repeated steps from 7/6 - neutral red, sampling, feeding, inoculating. **

Got larvae from Taylor shellfish 7/5/10. Larvae were spawned 6/30.
 * July 6, 2010**
 * __Cg Larvae - Vt trial 1__ **
 * 10:45 am **
 * salinity 31 ppt, T 20C, pH <8. Water flow is on for one hour every other hour. **
 * 11:20 am **
 * Put 50 mL of SW + larvae from each chamber into small glass vial and added 50 uL neutral red. Swirled gently and let sit 6 hours. **
 * 4:00 pm **
 * Emptied and cleaned all larval chambers. Refilled with fresh SW. Sampled larvae for RNA (1 per chamber). Replaced all non-stopper chambers with stoppered ones and replaced all banjo filters with clean ones. Fed larvae mix of T.iso and Plya (50 mL divided between 8 chambers). **
 * 5-5:30 pm **
 * Inoculated treatment chambers (A,B,D & E) with 100 uL heat-killed Vt (prepped by ED). **
 * Measured and assessed mortality of neutral red-dyed larvae from this am. Took 40 uL aliquots from each dyed sample and measured all larvae positioned well for measuring. If n<10 in the 40 uL aliquot, did a 2nd or 3rd aliquot. Noticed ciliate in chambers B & D. **


 * July 2, 2010**
 * __Cg Larvae__ **
 * Emptied and cleaned all larvae chambers. Sampled for RNA and microscopy as described below. Today was end of trial so cleaned out all chambers. Sizes recorded in spreadsheet. Live swimming larvae visible in all chambers except F. **


 * June 30, 2010**
 * __Cg Larvae__ **


 * Emptied and cleaned all larvae chambers. Replaced the banjo filters with new, clean 70 micron filters. Took one sample from each chamber to put in RNAlater and then a few larvae for microscopy. Stored the RNA samples in 500 uL RNAlater. Refilled all chambers with fresh seawater. **


 * Microscopy: Looked at all larvae under dissecting scope to assess if there were any still alive. All chambers except D & I had live larvae swimming around. Put 50 uL from each sample on depression slide and photographed under compound scope + camera to measure with SPOT. Measured larvae from all chambers except D & F because there were no larve in the aliquot. The only larvae measured were ones with a dorsal or ventral view that were providing a view of a circle (posterior or anterior views did not provide clear measurements). Measurements were taken along the longitudinal and latitudinal axes of the dorsal/ventral side of the larva. **


 * June 28, 2010**
 * __Cg Larvae__ **
 * made 1 L f/2 media for algae culture. See recipe 5/28. **


 * Emptied and cleaned all larvae chambers. Sampled RNA larvae for gene expression (in 500 uL RNAlater) and microscopy. For microscopy, just took samples from chambers A & F. Measured subset of these using microscope. Summary statistics (data are in spreadsheet CO2 Cg larvae sizes start 062310), measurements are in microns and letters indicate larval chamber: **

Under dissecting scope, larvae were observed swimming, although fewer were obviously active.
 * mean A || 100.4615385 || 91.23076923 ||
 * max A || 107 || 101 ||
 * min A || 87 || 78 ||
 * sd A || 4.875369808 || 7.037445632 ||
 * n A || 13 ||  ||
 * mean F || 101.25 || 96.75 ||
 * max F || 112 || 109 ||
 * min F || 84 || 83 ||
 * sd F || 8.091802811 || 7.967262562 ||
 * n F || 12 ||  ||

pH in treatment tank ~6, in control <8.

There appeared to be very few larvae left in chamber D but all chambers still had larvae.

Fed larvae Ply 429, 50 mL split between 8 chambers.

June 26, 2010 __Cg Larvae__ Drained and rinsed larvae from all chambers. Took samples for gene expression analysis and stored in 500 uL RNAlater. Looked at larvae from D & E under dissection microscope. Larvae are still alive and swimming vigorously. Chamber E seems to have many more larvae than the other chambers although good concentrations of larvae apparent in all chambers. Returned larvae to respective chambers and fed all chambers a mix of T.iso and Ply429 (50 mL between 8 chambers).

June 25, 2010 __Cg Larvae__ 12:30 pm pH in treatment tank <7, pH in control ~8 (measured with litmus paper) Changed pump cycle to 1 hour on/one hour off.

2:00 pm Put mixture of T.iso & Ply 429 in all chambers (dived 50 mL between 8 chambers).

June 24, 2010 __Cg larvae__ Got new larvae last night (6/23) from Taylor Shellfish. 5 day-old c. gigas. Put in chambers with new water around 5 pm with water flow and air bubbling. No co2 treatment.

Got new seawater from the aquarium today.

pH and pH probe: the pH probe is not properly calibrated to low pHs (it seems to be broken/missing a piece). It accurately measures the pH 7 standard, but not the 4. It is indicating that the seawater is at pH<7. After testing with litmus paper, it seems that the SW is probably at a pH of about 7. Turned down the pH probe so that it was about 0.2 units below what it thinks the seawater is so that CO2 would bubble into the treatment tank. There are now treatment and control chambers (same as before).

air: turned off air delivery to all chambers because the air flow was too strong.

water: put pumps on a timer so that they are on for one hour and off for 2. this is to make sure that the larval chambers do not overflow.

larvae sampling: filtered each larvae chamber on 64 micron mesh and rinsed in 2 buckets of water. Took sample for RNA analysis (0.5 mL RNAlater) and subsampled from 4 chambers for microscopy. Replaced larvae in chambers after rinsing and sampling with new water. Rinsed all chambers before putting larvae back. media type="custom" key="6449233" media type="custom" key="6449239" media type="custom" key="6449245"

June 19, 2010 __Cg Larvae__ Emptied and cleaned all larvae chambers. Took 1 sample from each chamber and stored in 0.5 mL RNAlater at 4C. pH for treatment was at 7.5. Also took a few larvae to observe in microscopy (from screen). There seemed to be fewer larvae in each chamber. Microscopy: a number of larvae were still alive and swimming vigorously. Under the dissecting scope, I did not see any evidence of ciliates. Larvae were put back in proper containers. We are low on seawater so I switched the system to recirculation, meaning that there are no longer treatment and control groups. media type="custom" key="6416959"

June 18, 2010 __Cg Larvae__ 6:20 pm, pH in treatment water 7.5. Treatment chambers are A, B, E, G. Controls are H, D, F, I. Sampled 2 mL of water from each chamber. No larvae were apparent in any of the aliquots. Will filter out all chambers tomorrow and thoroughly assess state of larvae.

June 17, 2010 __Cg Larvae__ 12:00 pm: lowered pH for water going to treatment chambers by about 0.4 pH units (to about 7.5). Made 900 mL F/2 media: 900 mL sterile seawater (from 5/28) 118.8 uL each Procul A & B (new bottles). Split algae cultures into new media: F/2 for T-iso and Ply429, F/2 + silicates for TW.

Cleaned all larvae in 2 washes seawater. While larvae on filter, removed >1000 and put in RNAlater for extraction. Took 2 samples from each chamber, stored at 4C. Before larvae put back in chambers, rinsed thoroughly with dechlorinated water and refilled with fresh seawater. Put some Ply429 in each of the chambers. At 8:00 pm the pH in the treatment water was down to about 7.3. In a number of the chambers with rubber stoppers (vs. resin) there were larvae stuck between the stopper and the side of the neck of the bottle.

June 16, 2010 __Cg Larvae__ about 8:30 pm. New larvae from Taylor (9 day-olds). ~89 larvae per mL in 1000 mL. Put 137.5 mL of larvae in each of 8 chambers - about 12,237 in 1.5 L. Chambers were filled with fresh seawater and attached to water and air supplies.

June 11, 2010 __Larval Chamber set-up__ Filtered out and washed all larvae. Put aside in 1 L beaker with seawater. Looked for ciliates in samples of water from tote and from water barrel, but found none. Dismantled and washed all parts of larval chamber set-up. New water set up and air flow with brand-new tubing. Holes in PVC done with 7/32 drill bit. media type="custom" key="6367383" media type="custom" key="6367389" media type="custom" key="6367391"

June 10, 2010 __Cg Larvae__ Counts per mL (based on 3 mL) A: 1 B: 16 H: 4 F: 1 E: 0 I: 1 G: 6 When present, most larvae were alive and many were swimming (veligers). Almost all larvae in chamber G were dead and there was one larger shell (2x size of others). media type="custom" key="6361117" __RNA Extraction__ Extracted RNA from larvae collected 6.9.10. Spun down tubes of larvae + RNAlater (5000xg for 5 minutes) and used pipette to remove larvae from bottom of tube. Extracted with TRI following manufacturer's protocol. Resuspended pellets in 50 uL water. All samples except F had visible pellets of RNA. Analyzed concentrations of RNA on Nanodrop. Stored samples at -80. (Letters indicate which larval chamber sample was taken from.) media type="custom" key="6360455"

__Where did all the larvae go?__ Emptied chamber A onto a 48 micron screen to capture all larvae in water column. Placed in bath of seawater, removed stopper and rinsed larvae stuck to stopper into petri dish. Rinsed & refilled A, replacing larvae on screen. Looked at stopper larvae under microscope and noticed that there were a number of ciliates in with the larvae (some larvae were still alive, but the ciliates were viciously attacking them). Looked at some of the algae from the hatchery under the scope but did not see ciliates. media type="custom" key="6361129" Emptied all other chambers, one at a time, onto a screen. Rinsed them twice in seawater on the screen. Emptied chamber completely, rinsed well, and refilled with fresh water. Replaced the water in the rest of the system as well. This process will need to be repeated daily to ensure expulsion of all ciliates.

June 9, 2010 __Cg Larvae__ Counted larvae in all chambers. Took 3 mL from each water column and counted # larvae per mL. Larvae were mostly alive and active except in chambers H & F where a large number were dead. Counts are in counts per mL, followed by average for the 3 mL and then estimated # per chamber. A: 21, 34, 52; avg. 35.7; total 53,500 B: 41, 15, 63; avg. 39.7; total 59,500 H: 10, 3, 12; avg. 8.3; total 12,500 F: 23, 25, 22; avg. 23.3; total 35,000 E: 0,0,1; avg. 0.3; total 450 I: 1, 4 (only 1 alive), 0; avg. 1.7; total 2,500 G: 22, 7, 25; avg. 18; total 27,000 media type="custom" key="6350337" media type="custom" key="6350339"

Sample ~1000 larvae from each chamber. Volumes will be as follows: A: 28 mL B: 25 mL  H: 120 mL  F: 43 mL  G: 55.5 mL  Volumes were filtered on a 48 micron mesh and larvae were removed from filter with a transfer pipette and immediately placed in ~0.5 mL RNAlater. Stored at 4C.

Changed water in system. Fed each chamber ~5 mL algae.

June 8, 2010

Went to Taylor Hatchery in Quilcene. Microbial Community Sampling (with Joth) ~11:00 am, Dabob Bay Sample depth: ~2m Used horizontal sampler to collect seawater from depth. Filled 2 plastic bottles (rinsed with seawater). Filtered 300 mL from each of these plastic bottles and fixed with 3 mL fixative. Folded papers and put in 2 mL tubes. Transported back to Seattle on ice and stored at -20C.

Also brought back 1 gallon algae mix for feeding larvae and 2 50 mL tubes with 8 day old C. gigas larvae (~488,000 larvae).

Counted larvae in chambers from last Thursday. There were none visible from the water column of any of the chambers. All the larvae had sunk to the bottom and were on/around the stoppers. Emptied out all old larvae and rinsed chambers.

Put new larvae in chambers. The following volume of evenly suspended larvae were put in each indicated chamber. A 100 mL B 400 mL D 100 mL E 100 mL F 100 mL G 100 mL H 100 mL I 100 mL This turns out to be 48,800 for the 100 mL chambers and 195,200 for B - or 33 larvae per mL and 130 larvae per mL, respectively. Put about 5 mL of algae mix in each chamber.

Partial water change in larval system. 1275937963
 * June 7, 2010**

Chamber E was emptied and F density reduced. Chambers A & B received 3 ml T-iso each (1 week old) Chambers C& H received 10 ml T-iso each (1 week old) Aquarium water postponed till next week. No feeding during weekend given supply. Will feed on Monday. Next week will also gear up for CO2 trials. Need to speak to Elene on how to preserve samples for pH measurements. 1275691235
 * June 4, 2010**


 * June 3, 2010**
 * __Cg Larvae__ **
 * __Notes on Feeding__ **
 * Ponis et al. 2003: **
 * in 2 L beakers (1.8 L seawater) **
 * D-hinge larvae at 5/mL **
 * feed 3x per week (when change sw) with 10^5 algae cells/mL **


 * Ponis et al. 2006 **
 * in 2 L beakers 5 D-hinge larvae/mL **
 * fed every other day, 50 cells/uL first week then 100 cells/uL **


 * __Quantifying Algal cells__ **
 * Took 20 uL from each algae culture flask. Looked at cells under scope - they were moving too rapidly to count accurately. Added ~45 uL Lugol's Iodine to each 20 uL aliquot (will multiply counts by 2.25 to make up for the dilution). Average number of cells per uL from each flask, with x2.25 correction, are as follows (counted on a hemacytometer): **
 * A1. Ply 429 5/28 w/foil top: 2.25 **
 * A2. Ply 429 6/1: 2.25 **
 * A3. Ply 429 5/28 w/ cotton: 3 **
 * B1. TW 5/28 w/ foil: 6.25 **
 * B2. TW 5/28 w/ cotton: 2.25 **
 * C1. T-iso 5/28 w/ foil: 218 **
 * C2. T-iso 5/28 w/ cotton: 422 **
 * C3. T-iso 6/1: 15 **


 * Aim to feed larvae 100 cells per uL of volume at which they are held. If feed equal amounts of each algae, will feed 33 cells/uL of each component. With 1.5 L (or 1.5 x 10^6 uL) in each chamber, need to feed ~4.95 x 10^7 cells of each algal component. **
 * Based on the cell counts above, the volumes from each of the flask to feed 4.95E7 cells would be: **
 * A1. 22 L **
 * A2. 22 L **
 * A3. 16.5 L **
 * B1. 7.9 L **
 * B2. 22 L **
 * C1. 227 mL **
 * C2. 117 mL **
 * C3. 3.3 L **


 * Brent brought back new C. gigas larvae from Taylor Shellfish in Quilcene. They are indicated at being 10 days old and when observed under microscope appear to be early veliger stage. Poured larvae into 1 L beaker of seawater. Plunged to evenly suspend larvae and sampled 10 mL. Counted 1 quadrant (1/4) of larvae in each mL (counts are estimates, not exact). The 10 1/4 counts are: 150, 60, 120, 50, 500, 50, 30, 45. This averages out to be about 380,400 larvae total or about 380 larvae per mL. **
 * There are 10 larvae per mL in chambers A & B. **
 * 50 larvae per mL in chambers C & H. **
 * The remaining larvae (approximately 527 mL worth, or 100,197 larvae) were evenly distributed between chambers E & F. This works out to be about 67 larvae per mL. **
 * All chambers are outfitted with air stones and new water delivered via drippers at 2 L/hour. **


 * June 2, 2010**
 * __Cg Larvae__ **
 * Replaced pump in larval chamber set-up. Measured pump rate (without drippers) at 420 mL/minute. **
 * Turned all drippers in chambers the other way and they are no longer having clogging problems. Looks like they were upside down before. **


 * May 29, 2010**
 * Fed Mac's oysters (top & bottom tanks) ~9am. **
 * Changed water in larval tank. Counted 5 mL larvae from chambers A, B and C. There were no larvae until the last mL (corresponding to water taken from the bottom of the chamber) for any of the samples. In A there were 32 larvae, 4 were alive and 2 were 2x the size of the others (dead). In B there were 8 larvae, 2 alive. In 7 there were 17 larvae, 1 alive. **


 * Extracted RNA from larvae samples collected 5.25.10 with TRI reagent following manufacturer's protcol. Homogenized larvae (even when none apparently visible) with pestle in TRI reagent. **


 * May 27, 2010**
 * __Algal starter cultures__ **
 * Sterilized 2 L of seawater in glass bottles (just sterilization cycle in autoclave). **
 * Sterilized varied flasks & graduated cylinders for growing algae on full sterilization through exhaust cycle. **


 * To make 1 L of pro-culture for algal starter cultures: **
 * Procul A & B = Kent Marine Pro-cultures A and B **
 * 10 mL Procul A + 10 mL Procul B for 20 gallons of water **
 * 1 L = 0.264 gallons **
 * 0.264 gallons * (10 mL/20 gallons) = .132 mL = 132 uL of Procul A & B for 1 L seawater **


 * Adding silicates: **
 * 1 gallon Procul A + 1 gallon Procul B makes 7,680 gallons culture water **
 * Add silicates at 20 g per 1,000 gallons of culture water **
 * 0.132 mL of Procul A + Procul B makes 38.425 gallons of culture water **
 * Need to add 0.7685 g of silicates **


 * Sterilized all equipment needed to make culture media in germicidal hood for 15 minutes. Made 1 L of regular culture media (Procul A & B) and 1 L of regular media + silicates. Media are made in sterile bottles, labeled, and at 4C (behind my bench). **


 * May 26, 2010 **
 * __Cg Larvae__ **
 * Changed water in system. **
 * Larvae counts for remaining chambers. Took 5 mL aliquots from each chamber, 1 mL in each well of 12-well plate. Counts are followed by observations **
 * A: 1 (dead), 3 (1 dead), 0, 38, 51 **
 * For the wells containing n=38 and n=51 only a few larvae were swimming in each well. In the n=38 well, 2 larvae were twice the size as the others (these 2 were alive, but not swimming so couldn't determine stage). All swimming larvae were veligers. **


 * B: 1, 0, 0, 1 (swimming), 2 (1 swimming) **
 * veligers **


 * C: 0, 0, 0, 0, 7 **
 * At least one larva was alive. None were swimming. **


 * May 25, 2010 **
 * __Cg Larvae__ **
 * Larvae counts for chambers - took 2 aliquots of 1 mL each and counted larvae under dissecting scope. **
 * A: n=2 & 4 **
 * B: n= 1 & 1 **
 * C: n=1 & 0 **
 * E: 0 **
 * Z: 0 **
 * All larvae were swimming actively (veliger). **
 * Noticed that there were what looked like larvae floating on the surface of the water and on the bottom of the holding tank. Took 1 mL aliquots of surface and bottom water. There were no larvae in the top sample but >50 from the bottom. **
 * Determined that larvae were escaping from Z & E. Filtered out all the water in these chambers through a 60 micron mesh and transferred to RNAlater. Took chambers into lab for repairs. **


 * May 24, 2010 **
 * __Cg Larvae: RNA & DNA Extraction__ **
 * RNA extraction : Following manufacturer's protocol for TRI Reagent. **
 * DNA extraction: manufacturer's protocol (details below) **
 * 1. Upon separation of sample into aqueous (RNA), inter-, and phenol phases, remove aqueous phase for RNA extraction. **
 * 2. Add 0.3 mL 100% EtOH to interphase + phenol phase and mix by inversion **
 * 3. store at RT 2-3 minutes **
 * 4. centrifuge 2000xg, 5 min, 4C **
 * 5. remove supernatant **
 * 6. wash DNA pellet 2 times. Each time wash with 1 mL 0.1 M trisodium citrate in 10% EtOH and store at RT for 30 minutes, periodically mixing. Centrifuge at 2000 xg for 5 min, 4-25C. **
 * 7. Suspend in 1.5-2 mL 75% EtOH. **
 * 8.store 10-20 min RT, mix **
 * 9. centrifuge 2000 xg 5 minutes, 4-25 C **
 * 10. remove EtOH and dry at RT **
 * 11. dissolve pellet in 0.3-0.6 mL 8mM NaOH **
 * 12. spin 12,000 xg 10 min **
 * 13. Remove the supernatant = DNA **


 * Reagents needed: 100% EtOH, 75% EtOH, 0.1 M trisodium citrate in 10% EtOH, 8 mM NaOH **
 * To make 0.1 M trisodium citrate in 10% EtOH: **
 * 2.941 g sodium citrate **
 * 90 mL H2O **
 * 10 mL 100% EtOH **

media type="custom" key="6217789"
 * The samples from 5.21.10 were the only ones with RNA and DNA pellets. **

__Cg Larvae__ Changed water in system. Sampled the following volumes (in mL) from the specified larval chambers - A & B: 5, 10, 20 C & E: 1, 2, 4 Z: 0.5, 1, 2 The volumes correspond to approximately n=50, 100, & 200 larvae, respectively.
 * May 23, 2010 **

Observations and counts of 1 mL of larvae from each of the larval chambers - A: n=9; actively swimming, veligers B: 0 C: n=4; actively swimming, veligers E: 0 Z: n=1; actively swimming, veligers

Spun down all samples at 1200 rpm for 5 min (20 C). Removed supernatant and stored at -80C.

__Cg Larvae__ Changed water in system.
 * May 22, 2010 **

__Cg Larvae__ Changed water in system. Joth brought in new larvae. Suspended in 1 L seawater, stirred well and aliquoted 1 mL 3 times into plate for counting larvae. Drew quadrants on the bottom of the wells. Counted larvae in 2 quadrants for each of the three aliquots. Per quadrant counts: I: 40, 52, 66 II: 70, 45, 90 Per well counts (quadrant * 4): I: 160, 208, 284 II: 280, 180, 360 average number of larvae per mL = 245 Total larvae = 245,000
 * May 21, 2010 **

Counts from chamber C (sampled 10 mL larvae + water): 180 larvae, 18 larvae/mL

For new larvae, want 2 chambers with 10 larvae/mL and 2 with 50 larvae/mL. 10 larvae per mL would = 15,000 larvae in the chamber (~1.5 L), which is 16.3 mL of the 245,000 larvae in 1 L. 50 larvae per mL would be 75,000 larvae per chamber, which is 81.5 mL of the 1 L. After aliquoting the larvae to their respective chambers, there is 804.4 mL of larvae + seawater left in the 1 L beaker. (Between each aliquot, plunged strainer up & down in beaker to suspend larvae homogeneously.) This 804.4 mL is about 197,000 larvae, or about 131 larvae per mL. Larval chambers with concentrations below: Chambers A & B: 10 larvae/mL Chambers C & E: 50 larvae/mL Chamber D: Elene's larvae (was chamber C) Foil-covered chamber (=chamber Z): 131 larvae/mL From chamber Z took 20 mL and 40 mL of larvae (this is about 26,000 and 52,000 larvae respectively). Spun down at 12000 rpm for 5 minutes (20C). Some larvae still in suspension after spinning down, but still removed most of seawater and froze remaining larvae at -80C.


 * May 19, 2010 **
 * __Cg Larvae__ **
 * Changed water in system. Last night Elene put many new larvae in chamber C. Counts today yielded about 55 live larvae in 10 mL for chamber C and almost not larvae in chambers B & D. New larvae arriving tomorrow as well. **
 * Empty out chambers B & D. Prepare chambers A, B, & D for larvae tomorrow. **


 * Sampling protocol for determining # of larvae needed to do usable RNA extraction. **
 * Every other day, sample 50, 100, and 200 larvae (10, 20, and 40 mL) from each chamber. Filter and preserve in RNAlater for RNA & DNA extraction. **


 * May 18, 2010 **
 * __Cg Larvae__ **
 * Changed water in system. Sampled 10mL from B and 9 mL from D. **
 * B 1/4 **
 * D 1/4 **


 * May 18, 2010 **
 * __Cg Larvae__ **
 * Changed water in system. Realized that pump for drip into chambers was not plugged in and so was not providing fresh water to chambers - plugged in pump. Sampled 10 mL from chambers B & D. **
 * B 7/26 **
 * D 24/57 **


 * May 17, 2010 **
 * __Cg Larvae__ **
 * Changed water in system. Sampled 10 mL from chambers B & D. **
 * B 5/20 **
 * D 6/8 **


 * May 16, 2010 **
 * __Cg Larvae__ **
 * Changed water in system. Sampled 10 mL from chambers B & D. **
 * B 9/18 **
 * D 0 **


 * May 15, 2010 **
 * __Cg Larvae__ **
 * Changed water in system. Sampled 10 mL from chambers B & D. //Counts are #live/total.// **


 * B 3/7 **
 * D 15/27 **


 * May 14, 2010 **
 * __Cg Larvae__ **
 * Suspect that did not transfer most of the larvae from holding chamber, that they were probably all on the very bottom. Put water drip & airstone in this larval chamber (now "D") under assumption that it contains larvae. **
 * Similarly to yesterday, took 10 mL from larval chambers B & D. For B sample, found only 1 dead larva. For D sample found 14 live & 58 dead. **


 * May 13, 2010 **
 * __Cg Larvae__ **
 * Brent got ~100 mL (~5000?) Cg larvae from Taylor yesterday. Put in larval chambers last night with bubbling air and water drip (chambers B & C). System is currently closed (i.e. not flow-through). **
 * Mortality estimates: **
 * Took 1 mL from top of larval chambers and then 2 aliquots of 1 mL from bottom of chambers B & C. Under dissecting scope, counted larvae alive & dead. **
 * Counts are #live/total larvae in aliquot **

Note: results may be inaccurate. During next mortality estimates, realized that ciliary action within/under larvae was present on most and was not noticed in previous observations. Took 10 mL from each larval chamber (started pipetting at bottom and then dragged pipette up through larval chamber so sampled entire water column). Aliquoted 1 mL into 10 wells of plate. Larval morphology: larvae are brown and transluscent (lighter brown inside). When moving have fringing beating cilia. When stationary, can see ciliary movement through larval body. Look similar to this media type="custom" key="6121565"
 * larval chamber || 1 || 2 || 3 ||
 * B || 0 || 0 || 0 ||
 * C || 0 || 0/8 || 1/4 ||
 * larval chamber || 1 || 2 || 3 || 4 || 5 || 6 || 7 || 8 || 9 || 10 ||
 * B || 1/1 || 0 || 0 || 0 || 0 || 0 || 0 || 0 || 0 || 3/3 ||
 * C || 0 || 0 || 0 || 0 || 0 || 0 || 0 || 0 || 0 || 0 ||

April 30, 2010 __Cg metal exposure__ Diluted cDNA 1:10 by adding 180 uL RNase-free water to each 20 uL sample. qPCR: EF1 (duplicate), metallothionein IV/MTIV, prostaglandin receptor/PE2. media type="custom" key="6008149"

April 28, 2010 __Cg metal exposure__ __DNase RNA__ Definite gDNA contamination in all RNA samples. DNase samples using Ambion's TURBO DNA-free rigorous protocol (see 3.18.10). media type="custom" key="5983801" __qPCR of DNased RNA with 18s primers__ Exact same PCR as 4.27.10. See sheet below for plate layout & mix recipe. Carry-over (or is it some contamination?) very much reduced. Data not meant for publication, so not going to worry about it. __RNA -> cDNA__ Reverse transcription of DNased RNA following protocol 11.6.09.

April 27, 2010 __Cg metal exposure__ __RNA Extraction__ Extracted RNA from gill tissues from both trials. Tissue IDs for 4.21.10 end date are: Cu1, Cu2, Cu3, C1, C2, C3. Tissue IDs for 4.26.10 end date are: Cu2.1, C2.1, C2.2, C2.3. RNA Extractions with Tri Reagent according to manufacturer's protocol. Resuspended in 50 uL RNAse-free H2O, heated for 5 min at 55C to dissolve. Concentrations of RNA measured on Nanodrop for each of the samples. media type="custom" key="5974143" __RNA -> cDNA__ Made cDNA from all RNA samples following protocol 11.6.09. Stored remaining RNA at -80 in C. gigas 2010 Box 1. plate layout:

Cu = copper trial C = control _2.# = 2nd trial media type="custom" key="5979187"
 * || 1 || 2 ||
 * A || Cu1 || C2.2 ||
 * B || Cu2 || C2.3 ||
 * C || Cu3 ||  ||
 * D || Cu2.1 ||  ||
 * E || C1 ||  ||
 * F || C2 ||  ||
 * G || C3 ||  ||
 * H || C2.1 ||  ||

Well that was silly, I forgot to check for gDNA carry-over. Maybe I did a perfect extraction and I have clean RNA. __qPCR of RNA with 18s primers__ media type="custom" key="5979077"

April 26, 2010 __Cg metal exposure__ 9:30 am End of trial Treatment: 2 mortalities, 1 live oyster at end of trial, silty green sediment at bottom of tank. Opened dead oysters for observation, took photos of one (below). Sampled gill tissue of live oyster (Cu2.1 4.26.10) and preserved in RNAlater. Shells of all 3 were bright green. Control: All 3 still alive, feces at bottom of tank. Sampled gill tissues of all 3 oysters and preserved in RNAlater.

media type="custom" key="5962167" media type="custom" key="5962175" media type="custom" key="5962181"

April 24, 2010 __Cg metal exposure__ 11:30 am Changed water in both tanks Treatment: murky green sediment at bottom of tank (silty). 1 mortality (stored at -20). To new tank water, added 5 mL of 0.4 g CuN2O6 dissolved in 20 mL sterile seawater. Also added 5 mL Shellfish Diet 1800. Control: Feces at bottom of tank. To new tank added 5 mL sterile seawater and shellfish diet. No mortalities.

April 23, 2010 __Cg metal exposure__ 10:00 am Treatment: oysters still alive.

April 22, 2010 __Cg metal exposure__ 10:40 am used juvenile Cg controls from previous experiment (n=7). Gave both treatment (n=4) and control (n=3) groups new seawater (1.5 L) and 5 mL Shellfish Diet 1800. Both tanks are aerated with air stones. Treatment: To tank added 5 mL of 0.4 g CuN2O6 dissolved in 20 mL sterile seawater. Control: Added 5 mL sterile seawater.

April 21, 2010 __Cg metal exposure__ 3:00 pm: end of treatment. Treatment: Removed oysters from treatment tank. 3 more mortalities (n=3 for sampling). Stored morts at -20. Opened 3 others that appeared to be alive and sampled gill tissue (stored in ~1.5 mL RNAlater, 4C). Control: Removed 3 oysters from control tank. Renewed water (no food) for remaining 7 (live!) oysters. Sampled controls as above with pictures of dissection. Saved sacrificed oysters for further analysis and comparison with morts from treatment at -20. media type="custom" key="5926459" media type="custom" key="5926465" media type="custom" key="5926469" media type="custom" key="5926473"

April 20, 2010 __Cg metal exposure__ 10:20 am Changed water in both treatment and control tanks. Gave both tanks 10 mL shellfish diet. Renewed metal in treatment tank, but at half of previous dosage (4.18.10 had diluted 0.39 g CuN2O6 in 20 mL sterile seawater, so added just 10 mL of solution to treatment and 10 mL sterile seawater to control). Observations control: water cleared of food added 4.18 with many dark feces at bottom. All alive. Observations treatment: water still murky from food, but some feces (many fewer than control) at bottom. 3 more mortalities (open shells). All shells still bright green. Stored morts at -20.

April 18, 2010 __Cg metal exposure__ 5:00 pm Changed water in both treatment and control tanks. Gave both tanks 10 mL new shellfish diet. Did not renew metal in treatment tank. Observations control: Water was clear and dark feces present at bottom of tank. All alive. Treatment tank: Water bluish and murky green with white sediment at the bottom. Oyster shells bright green. No apparent feces in tank. One mortality. Chose to suspend treatment for 2 days and then begin again with smaller dose. Stored mort at -20 for further dissection and analysis.

April 16, 2010 __Cg metal exposure__ 3:00 pm: Put juvenile Cg (n=10 each treatment) in 1.5 L seawater. Oysters are from North Bay,WA collected 4.3.10. Aerated water with airstones. To treatment tank (on right) added 1.4 uM copper (II) nitrate hydrate from Sigma Aldrich (0.394 g). 0.787 uM was shown to be sublethal environmentally realistic dose (Macey et al. 1999). CIIN was dissolved in 27.5 mL sterile seawater. To control tank added 27.5 mL sterile seawater. To both tanks added 7.5 mL Shellfish Diet 1800. Challenges will last 1 week. Water will be changed and metals replenished every second day.

April 15, 2010 __V. tubiashii culture__ Colony growth on plate from 4.14.10. Starter culture of 5 mL LB + 1% NaCL, 37 C 250 rpm at 8:45 am. 4:15 pm: starter culture is cloudy (bacterial growth). Inoculated ~100 mL 1x LB + 1%NaCl with starter culture and put in incubator at 37C, 250 rpm.

April 14, 2010 Make LB plates for Vt growth: Added 3 g bactoagar to 200 mL 1xLB + 1% NaCl. Mixed and put in autoclave, only sterilization cycle. Plated LB. Started culture of V. tubiashii on one of the plates to incubate overnight at 37C. (4:40 pm)

April 13, 2010 __V. tubiashii culture__ 2:20 pm: no growth in yesterday's starter culture.

April 12, 2010 __V. tubiashii culture__ 7:15 am: culture is still only a very little cloudy so will continue incubation. 5:45 pm: no growth in 100 mL culture. Re-inoculated starter culture (5 mL) with Vt from 4.8.10, left at 37 C 250 rpm.

April 11, 2010 __V. tubiashii culture__ Culture from 4.9.10 sat at RT over the weekend. At 8:00 am put at 4C. At 3:45 pm inoculated ~100 mL LB+1% NaCl. Incubated at 37 C 250 rpm.

April 9, 2010 __V. tubiashii culture__ 7:30 am: 5 mL culture no growth. Inoculated 5 mL LB + 1% NaCl, incubated at 37C 250 rpm. 2:45 pm: no growth in new culture. Inoculated ~2mL LB + 1% NaCl to see if less volume = more movement for cells = better growth (37 C, 250 rpm). Both cultures will be in incubator overnight.

__Oyster feeding__ Water was clear! Oysters ate! Lots of feces in bottom of tank. Replaced oysters in appropriate bags and put in large container. Renewed water & food in small tanks and put in Willapa Bay oysters (11 am).

April 8 2010 __V. tubiashii culture__ culture did not grow. Restreaked new LB plate + 1% NaCl with stock of V. tubiashii from -80 (frozen stocks box, bottom shelf, green eppie tube). Incubated plate at 37 C. Also started working culture in ~ 5mL LB medium + 1% NaCl at 37 C, 250 rpm around 10:30 am. 5:00 pm: Vt had grown on plate. Removed from incubator, sealed with parafilm, into fridge 4C.

__Oyster feeding__ Put 5 mL Shellfish Diet 1800 in 50 mL falcon tube. Gradually added 45 mL sterile seawater, mixing between additions to make 10% Shellfish Diet. Filled 2 small plastic tanks with ~ 2L seawater (the seawater that the oysters are kept in). In one tank, put n=21 juvenile Cg from NB (51x35, Ro15, from Dec. 2009) and ~10 mL 10% Shellfish Diet. In second tank, similar set-up but oysters were collected from North Bay 4.3.10. Water appears to be light green in both tanks (11 am).

April 7, 2010 __V. tubiashii culture__ Made 200 mL 1x LB + 1% NaCl: 40 mL 5x LB lab stock, 160 mL nanopure H2O, 2 g NaCl. Autoclaved without exhaust/dry cycle. Swiped Vt from plate grown 3.12.10 with sterile wand and swirled in ~100 mL LB + NaCL in flask. Put in incubator, 37C, 250 rpm overnight.

April 6, 2010 __FISH 310__ Ran 10 uL PCR products from 4.5.10 on 1.5% agarose gel + 10 uL EtBr.

April 5, 2010 __Vt-exposed juvenile Cg__ Determined concentration of samples on Nanodrop. media type="custom" key="5796031" Normalized cDNA concentrations to 68.6 ng/uL. qPCR of normalized cDNA for EF1. media type="custom" key="5799475"

__FISH 310__ PCR of student DNA with both Abalone and olympia oyster primers (positive and negative controls). Used Apex PCR mix: 12.5 uL Apex, 8.5 uL H20, 0.5 uL each primer; 22 uL master mix + 3 uL DNA. media type="custom" key="5799485"

March 30, 2010 __FISH 310 prep: species ID__ Prepared 100 mL 10 % chelex (100 mL nanopure water + 10 g chelex). Transferred to 3 falcon tubes. Used 20 uL pipette to aliquot 5 uL of EtOH + 1-4 larvae (verified presence of larvae in aliquot visually) into eppie tube. Only testing larvae B,C, & D since there are limited A & E. Left caps open to dry at RT in hood. Extracted DNA with chelex: aliquoted 300 uL 10% chelex into each tube w/ larvae. Heated at 100C for 20 minutes. Spun down and removed supernatant (DNA). Prepped master mix for abalone primer set & olympia oyster primer set according to lab worksheet - 19 uL MM, 1 uL template. PCR with each primer set for unknown DNA B,C,D, neg. chelex, neg. PCR and either positive abalone or oly depending on primer set. PCR'd at thermal profile in worksheet.

Prepared 1.5% agarose gel with EtBr. Ran 10 uL PCR product on gel with 100 bp ladder. Only positive amplification was positive controls for both species.

March 29, 2010 __qPCR of Vt-exposed juvenile oysters: complement C3 & complement receptor__ media type="custom" key="5753649"

March 26, 2010 __Primer reconstitution__ Reconstituted the following primers to 100 uM in TE buffer, pH = 8.0 (all for C. gigas): mannose binding, complement C3, properdin, complement receptor, ficolin, defensin, proline-rich polypeptide (prp), hemocyte defensin 2 (defh2), and BPI. All stored in primer box #6.

qPCR of new primers with cDNA. media type="custom" key="5738199" primers that failed: BPI, Prp, Defensin primers that amplified successfully: complement C3, complement receptor, mannose binding, properdin, ficolin. hemocyte defensin 2 warrants further testing. Below are the quantitation curves of the successful primers. media type="custom" key="5738201"

media type="custom" key="5728463"
 * March 25, 2010 **
 * __qPCR of Vt-exposed juvenile Cg: EF1 & prostaglandin receptor (PE2)__ **

media type="custom" key="5700807" media type="custom" key="5714473"
 * March 23, 2010 **
 * __qPCR of Vt-exposed juvenile Cg: IkB & IL-17__ **
 * Note of caution: These are non-normalized expression values. Normalization to come... **


 * March 22, 2010 **
 * __RNA -> cDNA__ **
 * Followed reverse transcription protocol on 11.6.09. **

media type="custom" key="5693119" media type="custom" key="5693127" media type="custom" key="5693131"
 * qPCR of cDNA with Hsp70, IkB, and IL-17. Negative and positive (gDNA) controls. **

media type="custom" key="5668865"
 * March 19, 2010 **
 * __Vt-exposed oyster reDNase__ **
 * Evidence of gDNA contamination from qPCR **


 * DNase with regular Ambion protocol. Used DNased RNA from 3.18.10. Added 1 uL DNase and 5 uL 10x TURBO buffer. Incubated at 37C for 30 minutes. **
 * Added 5 uL deactivation buffer & incubated at RT mixing 3 times. Spun at 10000xg for 1.5 minutes. Removed supernatant to clean tube and stored at -80. **

media type="custom" key="5693115"
 * qPCR of 1:20 dilution newly-DNased RNA with 18s primers (identical layout and protocol to 3.18.10). **

media type="custom" key="5661785"
 * March 18, 2010 **
 * __Vt-exposed oyster DNase__ **
 * Followed manufacturer's protocol (Ambion) for TURBO DNA-free, following the rigorous protocol. Made 10 ug in 50 uL dilutions of RNA. **
 * Added 0.5 uL DNase and 5 uL 10x TURBO buffer. Incubated at 37C for 30 minutes. Added 0.5 uL DNase and incubated 30 min at 37C. **
 * Added 5 uL DNase inactivation buffer to each sample, incubated for 2 min at RT and mixed 3x during incubation. Spun at 10000 xg for 1.5 minutes. Removed supernatant (RNA) to clean tube, stored at -80. **


 * __Make cDNA__ **
 * Followed protocol on 11.6.09. **


 * __qPCR__ **
 * qPCR of DNased RNA with 18s primers to test for gDNA contamination. **

media type="custom" key="5641507"
 * March 16, 2010 **
 * __Vt-exposed oyster RNA extraction__ **
 * Extracted all 16 gill samples (Cg+Vt 1-8 and CgC 1-8) with TRI Reagent following manufacturer's protocol. Resuspended dry RNA pellets in 100 uL ultrapure-superclean-RNase free water and heated at 50C for 5 minutes to put in solution. Measured concentration of samples on Nanodrop. **
 * Samples stored at -80C. **


 * March 15, 2010 **
 * __Oyster challenges__ **
 * Removed Vt from incubator at 1:50 pm. Took ~1 mL of bacteria + broth and spun down in centrifuge at max speed to make blank for measuring absorbance. **
 * Measured absorbance of cultured on Nanodrop at 620 nm to determine CFUs. **
 * CFU calculations are based on growth curve for V. tubiashii acquired from R. Elston. The CFU vs. absorbance trendline is described by the equation: y = 3E9x-3E8. **

For challenge, desired CFU is 10^4 per mL in 1.5 L seawater = 1.5E7 CFU total. This translates to 96.2 uL of bacteria culture in a 1.5 L seawater tank.
 * || ND absorbance at 620 nm || CFU ||
 * || 0.159 || 1.77E8 ||
 * || 0.147 || 1.41E8 ||
 * || 0.150 || 1.50E8 ||
 * average || 0.152 || 1.56E8 ||

Aliquoted 96.2 uL of bacteria in culture to a 1.5 mL eppie tube. Spun down for 2 minutes at RT, 5000 rpm. Removed supernatant (broth) and resuspended in 100 uL sterile seawater. Verification under microscope that vibrios were still alive (moving).

Took 1.5 L of seawater from oyster holding tank for each experimental set-up (5.5 L tanks) and put 8 juvenile oysters in each tank (Willapa Bay, 51x35). Provided air flow to each tank. In control group, aliquoted 100 uL sterile seawater. In experimental challenge aliquoted the resuspended vibrio. (3:30 pm). Challenge will last 3 hours.

6:30 pm. Brought experimental tanks of oysters into lab. Sampled gill tissue of all 16 oysters and put in labeled tubes of RNAlater. Sampled Vt-exposed oysters first. Between each oyster, all tools were sterilized first in 10% bleach and then in 95% EtOH + autoclaved sand. Oysters were shucked with a shucker and gill tissue was removed with forceps and scissors. Sample tubes were topped off with RNAlater (each tube contains 1+ mL RNAlater). Oyster shells seemed brittle and most oysters were pretty easy to open. After all samples were taken, tubes were put at 4C overnight. Stored at -80C.

March 14, 2010 __V. tubiashii culture__ In hood, swiped bacteria from plate with sterile scraper and swirled in ~55 mL LB broth + 1% NaCl in Erlenmeyer flask. Covered with foil and put in incubator at 250 rpm, 37C at 10:45 am.

March 12, 2010 __V. tubiashii plating__ Pre-made LB plate with 1% NaCl - incubate at RT for 15 minutes. Turn on UV light and blower in hood for 15 minutes. Vt stock from -80C freezer, kept on ice. Using sterile spreader, dipped spreader tip in frozen Vt and spread on plate. Incubate plate upside down at 37C until end of day (visible bacteria growth) and SW transferred to 4C.

February 16, 2010 __PCR of O. orca cDNA__ Cox2 did not amplify in the qPCR, but hsp6 amplified in all samples (same product size and only one product peak). Regular PCR of 3 samples (...) to verify amplicon size (primers were designed for hsp6 that amplifies at 523 bp in T. truncatus and at 350 bp in gray whale). template: 3 uL cDNA (1:10)


 * Reagent || volx1 || volx5 ||
 * 2xapex buffer || 12.5 uL || 62.5 uL ||
 * H2O || 8.5 || 42.5 ||
 * Pf || 0.5 || 2.5 ||
 * Pr || 0.5 || 2.5 ||

February 15, 2010 __O. orca Reverse Transcription & qPCR__ Followed RT protocol from 11.6.09, but used 10 uL of RNA and 6.75 uL of H2O with 0.5 uL oligo dTs. After final incubation, diluted entire cDNA 1:10 in 225 uL of H2O (stored in eppie tubes at -20C).

for qPCR, used Cetacea consensus primers for cox2 and hsp6 (primer box #4, F8 & F9, G1 & G2). Made dilutions to 10 uM and prepped master mix.

February 10, 2010 __O. orca RNA: Bioanalyzer 2100__ Based on concentrations from nanodrop 2.9.10, made 10 uL dilutions of all RNA samples to 5 ng/uL in DEPC water. media type="custom" key="5342015" Ran Pico chip on Agilent Bioanalyzer with herring RNA (sample 1) as control. In order, samples 2 through 11, ran RNA from L87, K13, L60, L57, L82, Luna, J19, L53, K27, L2. Results show probable RNA degradation of most of the biopsy samples, except for L53. media type="custom" key="5342033"

February 9, 2010 __RNA Extraction of Archival O. orca Blubber__

All samples are biopsies of blubber except for Luna and L60, which are samples taken from necropsies. L60 is a skin sample, Luna is a blubber sample. Extracted each sample with 1 mL TRI Reagent according to manufacturer's protocol. RNA pellets were resuspended in 50 uL DEPC water and incubated at 55 degrees C for 5 minutes. Samples were then mixed and nanodropped. Concentrations media type="custom" key="5339397"
 * Animal || Weight of Biopsy (g) || Fraction biopsy used || Weight tissue extracted ||
 * Luna || 0.1 || 1 || 0.1 ||
 * L60 || 0.1 || 1 || 0.1 ||
 * L57 || 0.09 || 1/2 || 0.045 ||
 * J19 || 0.1 || 1/2 || 0.05 ||
 * L73 || 0.09 || 1/2 || 0.045 ||
 * L87 || 0.05 || 1/4 || 0.0125 ||
 * K13 || 0.07 || 1/4 || 0.0175 ||
 * L53 || 0.1 || 1/4 || 0.025 ||
 * L2 || 0.08 || 1/2 || 0.04 ||
 * L82 || 0.05 || 1/2 || 0.025 ||
 * L67 || 0.08 || 1/4 || 0.02 ||
 * K27 || 0.06 || 1/4 || 0.015 ||

media type="custom" key="5199783" media type="custom" key="5199765"
 * January 20, 2010 **
 * __qPCR of IkB: CO2 challenged juvenile oysters__ **
 * All 16 samples in duplicate. gDNA for standard curve in dilutions 1:10, 100, 1000, 10000. **
 * January 19, 2010 **
 * __Primer test qPCR__ **
 * qPCR of new primer dilutions for matrilin, beta tubulin, and chaperonin. Included EF1 and Prx6 as controls. 2 samples with template and 4 negative controls. Still evidence of contamination in the new primer dilutions and in EF1 (not Prx6). Discarded working stocks of EF1, matrilin, beta tub., and chaperonin. **

media type="custom" key="5199769"
 * Test qPCR of IkB (with Prx6 as positive control). **

media type="custom" key="5165537"
 * January 13, 2010 **
 * __Contamination test of new primers, qPCR__ **
 * Before removing template from the freezer, made master mixes for primer sets EF1 (control), chaperonin, matrilin, and beta tubulin. 3 blanks for each primer were sealed. Template removed from freezer and 4 more wells (2 + template, 2 negative) were prepared for each set. **
 * PCR protocol as follows **

media type="custom" key="5148793" media type="custom" key="5150813"
 * January 12, 2010 **
 * __New primers qPCR__ **
 * Tested new primers with pooled cDNA (1 uL template per reaction). 2 wells of template, 4 wells of negative controls per primer set. **
 * Still evidence of contamination. Did another qPCR, new template and included Hsp70. Also included 2 wells in each primer column with just 25 uL of water. **


 * January 11, 2010 **
 * __New primers__ **
 * Ordered new primers for Cg, Beta tubulin, matrilin, and chaperonin. **
 * Made pH 8.5 TE Buffer: **
 * 1 mL Tris HCl 0.5M, pH = 6.8 **
 * 0.1 mL 0.5 M EDTA **
 * 48.9 mL nanopure H2O **
 * Brought pH up to 8.5 with 1N NaOH. **


 * Reconstituted dried primers to 100 uM using volumes based on 10Xnm. **

media type="custom" key="5106041"
 * January 4, 2010 **
 * __Contamination check__ **
 * Still evidence of contamination in PCR from 1.1.10. Ran PCR products on 1.5% agarose gel. Clear evidence of contamination for both primer sets on gel as well. **
 * Did qPCR of blanks withe primers Hsp70, Prx6, chaperonin, and matrilin. **


 * January 1, 2010 **
 * __qPCR optimization of matrilin and chaperonin primers__ **
 * temperature gradient of 54-66 degrees C. Used leftover pooled cDNA from differential display as template. Protocol outlined heremedia type="custom" key="5097227". **


 * December 31, 2009 **
 * __Contamination check__ **
 * Regular PCR, no template. Primers: beta tubulin, matrilin, chaperonin, Hsp70, and Prx6. Each reaction is 25 uL. **


 * Reagent || volx1 || volx5 ||
 * 2xApexBuffer || 12.5 uL || 62.5 ||
 * H2O || 11.5 || 57.5 ||
 * Pf || 0.5 || 2.5 ||
 * Pr || 0.5 || 2.5 ||

Ran PCR program CLNY under SBR menu on thermocycler. Plate layout below.

PCR with same primers, but added genomic DNA template (dilutions 1:100 and 1:1000). Used same master mix recipe, but 2 uL template per reaction and 9.5 uL water. Plate layout media type="custom" key="5095369" Ran PCR products on 1.5% agarose gel + EtBr at 100 V for ~45 minutes. media type="custom" key="5095701"

December 30, 2009 __Contamination check__ SW's PCR with primers was clean. I redid the qPCR with beta tubulin, matrilin, and chaperonin with and without template and with standards. Protocol media type="custom" key="5095245". Contamination still universal in negative controls.

December 29, 2009 __Contamination check__ previous qPCR's with DD gene discovery primers showed evidence of contamination. Suspect contamination was in water, including water in which primer dilutions were made. Made new 10 uM primer stocks with new water and did qPCR without template to compare old and new 10 uM primer dilutions for matrilin, beta tubulin, chaperonin, and TGF-b. PCR protocols media type="custom" key="5091831" Still evidence of contamination in the PCR. SW ran primers chaperonin, B tubulin, and matrilin with his master mix.

December 17, 2009 __qPCR with Gene Discovery Primers: CO2 challenged juvenile oysters__ Designed primers based on DD products (sequenced clones). From BLAST, 4 putative genes were identified: Beta tubulin, TGF-b, matrilin, and chaperonin. qPCR of B-tub. and matrilin as outlined media type="custom" key="5062345". Used standard curve of gDNA (0.445 ng/uL) at dilutions 1:10, 1:100, 1:1000, and 1:10000. In ng: 0.178-0.000178. qPCR of chaperonin and TGF-b as outlined media type="custom" key="5062341".

December 3, 2009 __Cloning of DD products: CO2 challenged juvenile oysters__ PCR of restreaked colonies Master Mix (x35) 2xApex buffer 875 uL water 805 uL  Pf 35 uL  Pr 35 uL  Aliquoted 50 uL of MM into plate wells. Touched sterile plastic wand to restreaked colonies and set in MM. Swirled wands in MM before removing. Program CLNY in SBR directory. media type="custom" key="4955421" Made 1.2% agarose gel and ran 20 uL of colony PCR product at 100V for ~30 minutes. media type="custom" key="4959575" Based on gel band sizes, did Qiagen minipreps for sequencing of the following samples (PS#-colony#): 10-4, 10-5, 10-6, 12a-1, 12a-3, 12a-5, 9-1, 9-4, 10-4, 10-5, 10-6, 12b-1, 12b-4, 18-1, 18-3, 38-2. Followed Qiagen MinPrep protocol to extract DNA from chosen cultured colonies. Poured ~1.5 mL of each LB+colony into minicentrifuge tubes. Spun at max speed, 2 min. Discarded supernatant and poured in 1.5 mL more, spun, and decanted (white pellet on bottom). Resuspended pelleted bacteria in 250 uL Buffer P1 via vortexgin. Added 250 uL Buffer P2 and mixed by inverting 4 times. Added 350 uL Buffer N3 and immediately mixed by inverting 4 times. Transferred supernatant to QIAprep spin column and centrifuged at 13000xg for 1 min. Washed column with 750 uL Buffer PE and spun at max speed for 1 min. Discarded flow through and spun additional minute at max speed. Put column in new eppie tube, added 30 uL Buffer EB. Let incubate at RT for 1 minute, then spun for 1 min. Put 10 uL of eluted DNA into plate to send off for sequencing.

December 2, 2009 __Cloning of DD products: CO2 challenged juvenile oysters__ Plates from 12.1.09 had few and faint colonies. Leave to incubate at 37C to allow for more colony growth. Grid a plate with 8 squares for PS#10 colonies and 8 squares for PS#12 (200 bp) and 2 squares for a negative control (dark blue colony) from each. Prepped master mix:

Aliquoted 50 uL master mix into wells of partial 96-well plate. Using sterile plastic wand, picked colonies, streaked them in the appropriate plate grid, and then placed wand (tip first) in corresponding MM-filled well in plate. Vol/color indicates the volume with which the plate was streaked (50 or 100 uL) and the color of the picked colony (white or light blue). Ran PCR on thermocycler, SBR directory, CLNY program. Thermal profile: 94C, 8:00 40 cycles: 94C, 45s; 50C 1:00; 72C, 1:30 72C, 10:00
 * Reagent || volx1 || volx18 ||
 * 2xApex buffer || 25 uL || 450 uL ||
 * water || 23 uL || 414 uL ||
 * Pf || 1 uL || 18 uL ||
 * Pr || 1 uL || 18 uL ||
 * Well || Sample || vol/color || Well || Sample || vol/color ||
 * A1 || 10-1 || 50/W || A2 || 12-1 || 50/W ||
 * B1 || 10-2 || 50/W || B2 || 12-2 || 50/B ||
 * C1 || 10-3 || 50/W || C2 || 12-3 || 50/W ||
 * D1 || 10-4 || 50/W || D2 || 12-4 || 100/W ||
 * E1 || 10-5 || 100/W || E2 || 12-5 || 100/B ||
 * F1 || 10-6 || 100/W || F2 || 12-6 || 100/B ||
 * G1 || 10-7 || 100/W || G2 || 12-7 || 100/B ||
 * H1 || 10-8 || 100/W || H2 || 12-8 || 100/W ||

Made 1.2% agarose gel (1.2 g agar, 100 mL 1xTAE, 10 uL EtBr). Loaded 20 uL 100 bp ladder and PCR product. Ran at 100 V for ~45 minutes. Gel 1: ladder A1 B1 C1 D1 E1 F1 G1 H1 A2 B2 C2 D2 E2 F2 G2 media type="custom" key="4955247"

Gel 2: ladder H2 A3 B3 media type="custom" key="4955253"

Restreaked and seeded all colonies grown on 12.1.09 (see table below). Made 200 mL LB+Kan: 50 mL 5x lab stock + 160 mL nanopure H2O + 200 uL Kan. Aliquoted 5 mL of LB mix. to glass tubes with sponge stoppers. Touched a sterilized toothpick to each colony and dropped into growth medium. Based on gel results above, did the same for chosen colonies of PS#10 and PS#12a that appeared to have different products: 10-4, 10-5, 10-6, 12a-1, 12a-3, 12a-5. Grew up overnight at 37C, 250 rpm. Did not exceed 8 colonies for any primer pair. media type="custom" key="4955351"

December 1, 2009 __Cloning of DD products: CO2 challenged juvenile oysters__ Repeated procedure from 11.30.09 for primer sets 1, 9, 12 (400 bp), 18, 38 and 57. The following changes were made to the procedure: Cloning reaction was cut in half so only 2 uL PCR product, 0.5 uL salt solution and 0.5 uL vector were used. LB+Kan plates were streaked with volumes of either 50 uL or 200 uL competent, transformed cells. Plates were streaked around 3 pm and left to incubate overnight at 37C.

November 30, 2009 __Cloning of DD products: CO2 challenged juvenile oysters__ Used TOPO TA cloning kit (Invitrogen), T10 competent cells. PCR products to clone: primer st #10 (11.19.09), PS#12 (11.17.09) 200 bp band Let bands thaw at RT. Spun at 5000 g for 10 min (RT) in ultrafree-DA tubes. For cloning reaction in strip tubes: 4 uL PCR product 1 uL salt solution (TOPO kit) 0.9 uL vector (TOPO) put on thermocycler for 10 minutes at 22C, then directly to ice. Added 2 uL of each cloning reaction to a tube of competent cells (thawed on ice right before use); swirled as added. Incubated on ice for 10 minutes then heat shock in 42C water bath for 30 s. Put on ice for 2 minutes. Added 250 uL room temp SOC medium to competent cells + cloning reaction. Rolled tubes to coat sides. Put in 37C incubator at 225 rpm for 1 hour. Warmed LB+Kan plates to RT (4 plates total). Spread with 80 uL 20mg/mL Xgal. Dried plates at 37C. Plated out transformed cells, for each primer set plated 1 plate with 50 uL and one with 100 uL. Incubated at 37C with lids cracked to dry residual liquid and then upside down overnight.

Blue and white colonies appeared on all 4 plates. Colonies picked and restreaked on 12.2.09.

November 25, 2009 __qPCR of CO2 challenged juvenile oysters__ qPCR in duplicate of [|EF1, HIF], [|MDR, and Prx6]. Used cDNA from 11.6.09.

November 24, 2009 __Differential Display: CO2 challenged juvenile oysters__ Loaded PCR product for primer sets 56, 57, 58 (7 uL, 20 uL 100 bp ladder) on 2 % agarose gel, run at 100V for ~45 minutes. Imaged with UV. Differential expression (high MW) in CO2 pool at primer set 57 (even though you can't see it, it is there! I'm just terrible at taking gel photos). Cut out band and stored at -20. media type="custom" key="4911961" __Cloning of DD products__ Diluted 100 mL 5xLB (lab stock) with 400 mL nanopure water in large flask. Added 7.5 g Bacto Agar. Put in autoclave (121C, 20 minutes). After autoclaving, let LB cool until flask is comfortable to touch for at least 30s. Added 500 uL Kanamycin (50 ug/mL). Swirled flask until homogenized. Poured LB+kan into sterile plates with lids until LB covered bottom. Let sit ~30 minutes til set. Stored in fridge.

November 23, 2009 __Differential Display: CO2 challenged juvenile oystersmedia type="custom" key="4883315"__ Gene fishing (SeeGene) PCR with 3 new primer sets: 56, 57, 58. There are no new primer sets left so will have to use Sigma kit if to continue gene discovery.

November 20, 2009 __Differential Display: CO2 challenged juvenile oysters__ New Gene fishing PCR with 7 new primers (same protocol as previously): 1, 16, 34, 35, 37, 39, 40. Plate layout:

1258949412please start embedding images as this is the only way we can backup notebooks- thanks
 * A || p1CO2 || p1cont ||
 * B || p16CO2 || p16cont ||
 * C || p34CO2 || p34cont ||
 * D || p35CO2 || p35cont ||
 * E || p37CO2 || p37cont ||
 * F || p39CO2 || p39cont ||
 * G || p40CO2 || p40cont ||
 * H ||  ||   ||

2% gel with 100 mL 1xTAE, 2 g agar, 10 uL EtBr. Loaded 7 uL of each sample in order of primer pairs (1-40), control loaded first and CO2 loaded second. [|Differential display] of high MW band in primer set #1 control (gene is downregulated in CO2 group). Cut out band and stored in 1.5 mL eppie at -20C. media type="custom" key="4911987"

November 19, 2009 __Differential Display: CO2 challenged juvenile oysters__ Made 2% agarose gel: 100 mL 1xTAE, 2 g agar, 20 uL EtBr (oops!). Loaded gel as follows (7 uL of each sample) and ran at 100 V for ~45 minutes. (20 uL 100 bp Ladder on far left)

Differentially expressed [|band] in CO2 cDNA at primer set 10. Cut out and stored at -20. media type="custom" key="4911945"
 * p3cont || p3CO2 || p4cont || p4CO2 || p5cont || p5CO2 || p8cont || p8CO2 || p10cont || p10CO2 || p11cont || p11CO2 || p30cont || p30CO2 || p33cont || p33CO2 ||  ||

November 18, 2009 __Differential Display: CO2 challenged juvenile oysters__ Same procedure as 11.13.09 and 11.12.09. 8 new primers:3,4,5,8,10,11,30,33. PCR run on 96-well MJ thermocycler. Program called "DF".

Plate layout:


 * A || p3CO2 || p3cont ||
 * B || p4CO2 || p4cont ||
 * C || p5CO2 || p5cont ||
 * D || p8CO2 || p8cont ||
 * E || p10CO2 || p10cont ||
 * F || p11CO2 || p11cont ||
 * G || p30CO2 || p30cont ||
 * H || p33CO2 || p33cont ||

November 17, 2009 __Differential Display: CO2 challenged juvenile oysters__ Rony Thi re-ran previous DD samples (11.13 and 11.12) with 7 uL product per well. See her notebook for layout. Gel image [|here] media type="custom" key="4911935"

November 13, 2009 __Differential Display: CO2 challenged juvenile oysters__ Made 2% agarose gel (100 mL 1x TAE + 2 g agar) with 10 uL EtBr. Loaded 27.5 uL 100 bp ladder in leftmost well. Loaded samples, paired treatment and control, in primer pairs. Gel layout as follows: ladder p12CO2 p12cont blank p17CO2 p17cont blank p18CO2 p18cont blank p20CO2 p20cont Ran [|gel] at 100 V for ~45 minutes.media type="custom" key="4911907"

Possible differential display of high MW band for primer 18.

PCR of 8 new primers:6,7,9,13,14,15,19,38. Follow protocol from 11.12.09. Plate layout is:


 * A || CO2p6 || contp6 ||
 * B || CO2p7 || contp7 ||
 * C || CO2p9 || contp9 ||
 * D || CO2p13 || contp13 ||
 * E || CO2p14 || contp14 ||
 * F || CO2p15 || contp15 ||
 * G || CO2p19 || contp19 ||
 * H || CO2p38 || contp38 ||

November 12, 2009 __DIfferential Display: CO2 challenged juvenile oysters__ Resuspended pooled RNA in 18 uL water. Heated at 55 degrees C for 5 minutes. 'Speced pooled RNA on Nanodrop. Pool 1 was measured at 169.0, 170,3 and 172.3 ng/uL for an average of 170.53 ng/uL (3.08 ug) and Pool 2 was 26 ng/uL. Repeat pooling and RNA precipitation for Pool 2. Resuspend in 9.5 uL DEPC H2O. Concentration on Nanodrop measured at 737.9 ng/uL and 745.4 ng/uL (average of 741.65 ng/uL, or .74165 ug/uL). Following manufacturer's protocol for SeeGene GeneFishing, need up to 3 ug pooled RNA per reaction, no more than 7.5 uL. 7.5 uL of Pool 1 is 1.3 ug. 1.3 ug of Pool 2 is 1.75 uL. RT reaction for Pool1: 7.5 uL RNA + 2 uL 10 uM dT-ACP1. RT reaction for Pool2: 1.75 uL RNA + 2 uL 10 uM dT-ACP1 + 5.75 uL water Incubated tubes at 80 degrees for 3 minutes then to ice for 2 minutes. Added to each tube: 4 uL RT 5x MMLV buffer, 4 uL 2.5 mM dNTPs, 1.5 uL water, 1 uL reverse transcriptase. Incubated on thermocycler 42 degrees for 90 minutes then 94 degrees for 2 minutes. Chilled for 2 minutes on ice and added 80 uL water to each tube. Vortexed to mix.

For PCR, added reagents to wells of plate (8 wells) according to following: 3 uL cDNA 2 uL 5mM arbitrary primer 1 uL 10 uM dT-ACP2 4 uL distilled water 10 uL SeeAmp 2x master mix Plate layout as follows:

Placed on preheated (94C) thermocycler and started program: 94C 5 min; 50C 3 min; 72C 1 min; 40 cycles 94C 40s, 65C 40s, 72C 40s; 72C 5 min.
 * A || p12-CO2 ||
 * B || p17-CO2 ||
 * C || p18-CO2 ||
 * D || p20-CO2 ||
 * E || p12-cont ||
 * F || p17-cont ||
 * G || p18-cont ||
 * H || p20-cont ||

Stored finished PCR at -20 (and leftover cDNA).

November 11, 2009 __Differential Display: CO2 challenged juvenile oysters__ The following protocol basically follows Sigma's Differential Display manufacturer's protocol. I. Nanodrop RNA samples (these samples are the 2x DNased) [|Spec] (2 [|sheets] ) each sample (of 16) 3 times, average concentration for final concentration. Base subsequent calculations on this final concentration. Pooling samples: Amount of RNA per pooled sample cannot exceed 8 ug or 18 uL. To determine amount of each sample that will go in to pool, set up equation in Excel so that as sample with smallest concentration's volume was increased, all other sample volumes would commensurately increase as well. Since total volumes ended up being larger than 18 uL, need to precipitate RNA and resuspend in appropriate volume. Pool 1 (CO2 challenged) Pool 2 (controls)

RNA Precipitation: To each pool of RNA, add 0.1 volume 5 M ammonium acetate (9.36 for Pool 1 and 7.63 for Pool 2). Added 2 volumes 100% EtOH (205.9 uL and 167.88 uL, respectively) to each pooled RNA. Mixed well and incubated at -80 for 30 minutes. Spin tube max speed (16,000xg) at 4 C for 30 minutes. Discard supernatant and quick spin tubes to remove residual EtOH. Add 1 mL 70% EtOH; flick to get pellet off bottom of tube. Spin at max speed, 4C for 10 minutes. Discard supernatant. Remove residual EtOH. Stored at -80.

November 9, 2009 __RT qPCR of CO2 challenged oyster cDNA__ Repeated qPCR of previously amplified HIF and MDR, in duplicate, to ensure consistency of results. Master mix and cycling parameters for PCR detailed [|here].

November 6, 2009 __Reverse Transcription of CO2 challenged oyster RNA (take 2)__ Adapted protocol for MMLV Reverse Transcription. Reactions are 25 uL, approximately 1 ug total RNA per reaction. Added 1 uL from each sample, CO2 challenged (n=8) and control (n=8), to well in partial PCR plate. To each sample, added 16.75 uL ultra-clean water and 0.5 uL Oligo dT primers. Heated samples at 70 degrees C for 5 minutes (main directory on thermocycler, 75FOR5). Transferred samples immediately to ice for <10 minutes. Prepared master mix (for 17 reactions):

Mixed well and added 6.75 uL to each RNA sample. Mixed, spun down and incubated at 42 degrees for 1 hour followed by 3 minutes deactivation at 95 degrees C (REVTINC).
 * Reagent ||  ||
 * volx1 || volx17 ||
 * 5x MMLV Buffer 5 uL || 85 uL ||
 * 10 mM dNTPs 1.25 uL || 21.25 uL ||
 * M-MLV RT 0.5 uL || 8.5 uL ||

RT-qPCR Diluted cDNA 1:10 by putting entire cDNA reaction sample in 225 uL of pure H2O (eppie tubes, stored at -20). Prepared master mix and did qPCR at EF1 and Prx6 in duplicate according to [|here].

November 3, 2009 __RT-qPCR on Vibrio & CO2 challenged oyster cDNA__ [|PCR] on genes MTIV, hsp70, Prx6, and HIF1a.

November 2, 2009 __RT-qPCR on Vibrio & CO2 challenged oyster cDNA__ Same as 10/29/2009 but with new primers MDR, SOD, and AURKA. No positive control. See [|here] for layout, etc.

October 29, 2009 __RT-qPCR on Vibrio & CO2 challenged oyster cDNA__ Did RT-qPCR on all 16 gigas samples for genes elongation factor 1, Interleukin 17 Isoform D, and Cytochrome P450. Working stock primers were all prepared fresh: 90 uL H2O + 10 uL 100 uM stock to make 10 uM solution. Positive control C. gigas cDNA was provided by SW. Experimental cDNA samples were brought up to 80 uL (1:4 dilution) with 60 uL H2O. Master mix and PCR performed according to [|here].

October 28, 2009 __Vibrio & CO2 challenged oysters: test for contamination__ gDNA contamination apparent in all samples and blank controls. Made new 10 uM stock 18s gigas primers and got new aliquot of PCR water. Ran PCR of blanks and positive controls to ensure no contamination according ([|here] ). Results: blanks were clean, gDNA controls amplified as expected.

qPCR of 2xDNased 1:20 dilutions from 10/27/09 as seen [|here].

gDNA contamination gone from samples (proper amplification of positive controls, empty negative controls). Performed reverse transcription on RNA samples (undiluted 2xDNased). Transferred 5 uL of each sample into strip tube. Incubated at 75 degrees C for 5 minutes. Transferred to ice for 5+ minutes. Prepared master mix (x18): 72 uL 5x AMV RT buffer 144 uL 10 mM dNTP mix 18 uL AMV RTranscriptase 18 uL Oligo dT Primer 18 uL RNase free water Aliquot 15 uL of master mix into each sample well. Incubated for 10 minutes at RT. Incubated at 37 degrees for 1 hour then at 95 degrees for 3 minutes. Store at -20.

October 27, 2009 __Vibrio & CO2 challenged oysters: gDNA clean-up (x2)__ Still detectable gDNA contamination in PCR. Repeated clean-up protocol from Oct. 23, but performed protocol on samples that were cleaned once already. Since 5 uL had been removed from each of these to make the 1:20 dilution for the PCR, added only 4.5 uL TURBO buffer to the RNA. Prepped 1:20 dilutions for RT PCR to check genomic contamination. Discarded previous dilutions so all DNased samples and dilutions have undergone 2 rounds of DNasing.

Repeated PCR identical to 10/26 with 2xDNased RNA (same layout, master mix, and cycling parameters). See sheet from 10/26/09.

October 26, 2009 __Vibrio & CO2 challenged oysters: gDNA clean-up__ Still evidence of contamination in Dnased samples from 10.23, but SW and MG say that is probably only detectable because did not dilute RNA template enough before PCR. Repeated protocol from 10.23 but used 5 uL of 1:20 dilutions of DNased RNA template. Master mix, plate layout, and cycling parameters [|here].

October 23, 2009 __Vibrio & CO2 challenged oysters: gDNA clean-up__ For each RNA sample, added either 10 or 5 uL RNA to 40 or 45 uL water. This was determined based on concentrations from the Nanodrop (see 10.7 and 10.15). Samples with concentrations >1 ug/mL were diluted 5 uL RNA in 45 uL water; samples with concentrations <1ug/mL were diluted 10 uL RNA in 40 uL water. The 10 uL samples were CO2 juv 1, CO2 juv 5, CO2 juv6, and cont juv 7. The rest were diluted 5 uL in 45 uL water. (All in 0.5 mL tubes.) To the 50 uL diluted RNA, added 5 uL 10x TURBO Buffer (Ambion TURBO DNA free kit) and 1 uL TURBO DNase (2U). Mixed gently and spun down. Incubated samples at 37 degrees C water bath for 30 minutes. Added 5 uL DNase Inactivation Reagent to each tube (before adding, vortexed DIR to resuspend). Vortexed samples with DIR. Incubated at room temp for 2 minutes, vortexing twice during incubation. Centrifuged samples at 10000 xg for 1.5 minutes. Transferred RNA (supernatant) to clean tubes labeled with name of sample, "RNA", and "gDNA-free", date, and initials. After PCR, stored cleaned RNA at -80. Prepared qPCR master mix with 18s gigas primers and performed qPCR following previous protocol as outlined [|here]. (Only change is used 1 uL template instead of 5 uL, and 24 uL master mix per reaction.)

October 15, 2009 __Vibrio & CO2 challenged oysters: Nanodrop & RT-PCR__ Measured concentration of RNA extractions from 10.14.09 on [|Nanodrop]. NB: second "control-1" is really control-2.

Prepared master mix and ran RT-PCR as outlined [|here].

October 14, 2009 __Vibrio & CO2 challenged oysters: RNA Extraction__ Extracted from remaining whole body samples from TG's experiment. (See Oct. 7) Tissue weights and 1/2 TriReagent volume: CO2 juv 1 0.29 g 1.5 mL Tri CO2 juv 2 0.28 g 1.5 mL Tri CO2 juv 3 0.56 g 3 mL Tri CO2 juv 4 0.22 g 1.25 mL Tri CO2 juv 7 0.53 g 2.75 mL Tri CO2 juv 8 0.20 g 1 mL Tri cont juv 1 0.37 g 2 mL Tri cont juv 2 0.33 g 1.75 mL Tri cont juv 3 0.38 g 2 mL Tri cont juv 4 0.48 g 2.5 mL Tri cont juv 5 0.37 g 2 mL Tri cont juv 6 0.88 g 4.5 mL Tri

Tissues were homogenized using sonicator in volume of TriReagent indicated above. 500 uL of homogenate and 500 uL of fresh Tri were then combined in eppie tub and mixed thoroughly. Remaining tissue homogenized in 1/2 Tri was stored at -80 in 15 mL Falcon tubes. RNA was extracted following manufacturer's protocol. Pellets are a grayish-brown color but solubilized easily in water.

October 12, 2009 __RT-PCR of oyster RNA to determine genomic contamination__ RNA from 10.7.09 PCR consists of 4 experimental samples (CO2 juv 5, CO2 juv 6, control juv 7, and control juv 8), 4 negative controls of 5 uL water instead of template, 1 negative control of just master mix, and 3 positive controls of genomic oyster DNA at different dilutions (1:10, 1:100, 1:1000). Prepared master mix and ran RT PCR as outlined in [|data sheet].

October 7, 2009 __Vibrio & CO2 challenged oysters: RNA Extraction__ Selected 4 juvenile whole body samples from Tim's experiment. 2 were challenged with 970 ppm CO2 and 2 were controls. Tissue weights: CO2 juv 5 0.17g 1 mL Tri CO2 juv 6 0.38 g 2 mL Tri control juv 7 0.16g 1 mL Tri control juv 8 0.86g 4.5 mL Tri Added 1/2 suggested Tri Reagent to each sample (volumes next to weights) and homogenized with tissue sonicator. Added 0.5 mL of homogenized tissue in 1/2 Tri to 0.5 mL Tri for 1 mL volume samples. Stored remaining tissue in 1/2 Tri in 15 mL Falcon tubes in -80. Followed manufacturer's protocol for remaining RNA extraction. During first spin (10000 rpm for 15 min at 4 degrees C) CO2 juv 5 and control juv 7 tubes shattered in rotor tubes. Removed samples from rotor, put in new falcon tubes and respun (will now be one step behind CO2 juv 6 and control juv 8). Continued in protocol for samples 6 & 8 (isopropanol step). 5 & 7 pellets are black, so probably no good but will continue extraction anyway. Afters spec'-ing samples on Nanodrop, stored in -80. Labeled eppie tubes with sample name (as above), date, and RNA.


 * RNA concentration on Nanodrop 10.7.09 ||  ||
 * Sample ID || ng/uL || A260 || A280 || 260/280 ||
 * CO25 || 294.4 || 7.36 || 3.7 || 1.99 ||
 * CO26 || 565.61 || 14.14 || 7.212 || 2.34 ||
 * Cont7 || 650.46 || 16.262 || 7.893 || 2.06 ||
 * Cont8 || 2194.82 || 54.87 || 27.557 || 1.95 ||

September 21, 2009 __Salmon Senescence: RNA Extraction__ Selected 4 brain samples for extraction: 21, 23, 24, and 27 Brain weights:
 * 1) 21 0.47g
 * 2) 23 0.48 g
 * 3) 24 0.39 g
 * 4) 27 0.51 g

(For the most part I followed the manufacturer's protocol.) Cleaned homogenizers before use. Transferred all tissue to 5 mL Tri Reagent (this is half the amount of total reagent that should be used for this amount of tissue). Homogenized tissue twice. Transferred 3.5 mL of the homogenate to 3.5 mL of fresh Tri Reagent in clean tubes (remaining homogenate was stored in 50 mL Falcon tubes at -80). Thoroughly vortexed tubes and let sit for 5 minutes at room temperature. Added 1.4 mL chloroform to 7 mL of Tri Reagent mixture. Let sit for 15 minutes at room temp. Put 3.5 mL isopropanol in a clean tube and added aqueous (top) phase of Tri-mixture. Sample 27 had the least amount of aqueous phase and a very large interphase. Let sit for 10 min at room temp and centrifuged 12000xg for 8 min (SR did this part). Decanted/removed all isopropanol and washed remaining pellet with 7 mL 75% EtOH. Centrifuged 5 min at 10000 rpm. Removed EtOH with pipet and quickly centrifuged. Removed remaining EtOH with pipet and kimwipe. Let tubes air dry in hood for 5 minutes. All were dry except for 27. Resuspended 21, 23, & 24 in 50 uL nuclease-free water (27 was left to dry longer). Measured RNA concentrations on the nanodrop (the "view report" function wasn't working so the following are approximate results): 27 was not completely dry, but consulted Sam and resuspended in 50 uL water followed by heating in water bath at ~55 degrees for 5 minutes. Its concentration was 176 ng/uL. Stored extracted RNA in -80 in box labeled "Salmon Senescence".
 * 1) 21 ~1600 ng/uL
 * 2) 23 ~1400 ng/uL
 * 3) 24 ~1400 ng/uL