Lab+2

Daniel Bascom December 3, 2011 FISH 411


 * Lab Reflection**

I believe I got the results I did because at some point my samples were left out too above -20 degrees Celsius causing our HSP 70 to denature. It may have also been a result of leaving our gel running for too long at 150 V, going for 45 minutes instead of 20. user:emmatsbiological explanations, please!

My two main obstacles came from not having enough time to run through every step of the SDS-PAGE and the Western Breeze on my own. In two occasions I had to either leave early for a meeting or couldn't finish it the next morning because of class. Other people who we're involved on those gels were able to cover the difference, but its nice to be present for it the whole the through. I was also unable to run all the samples I wanted because of our limited resources, since many of us we're testing for the same thing we could share a gel and the work, but it limited the number of samples we could do in one try.

When doing science or any technical task its important to have intimate knowledge of all the steps involved to avoid errors. The project was a good example of what happens when there are even slight mistakes, they can compound and make it difficult to determine when and where things went wrong. I would say it is important because everything is important and everything is connected to everything. If it wasn't important then I wouldn't have done it and they wouldn't be asking me about it. user:emmatsInteresting logic, but please go a little deeper and give a real explanation.

Where did I go wrong? Was it during the protein extraction, when we ran the Gel, when we did our wash? I could compare my results to what people doing HSP RNA analysis got, if they didn't find HSP 70, but maybe another HSP, it would confirm my results.

//Hamdoun, Amro M, Daniel P. Cheney, and Gary N. Cherr. "Phenotypic Plasticity of Hsp70 and Hsp70 Gene Expression in the Pacific Oyster (crassostrea Gigas): Implications for Thermal Limits and Induction of Thermal Tolerance." Biological Bulletin. 205.2 (2003): 160-169. Print.//

Change in temperatures causes differences in the expression of HSP proteins 70, 72, and 77. Its possible I had binding in all of my samples because different HSPs were expressed in all and our antibody was not specific enough to target just one. Differences in pH, like temperature, could elicit a plastic response in HSP proteins.

//Boutet, Isabelle, Arnaud Tanguy, Sabrina Rousseau, Michel Auffret, and Dario Moraga. "Molecular Identification and Expression of Heat Shock Cognate 70 (hsc70) and Heat Shock Protein 70 (hsp70) Genes in the Pacific Oyster crassostrea Gigas." Cell Stress Chaperones. 8.1 (2003): 76-85. Print.//

This paper discusses the genes responsible for HSP, and might give me some insight into what my antibodies did target. It also discusses how HSP is translated from RNA, and what factors can affect this process. Has some interesting info on how oysters respond to heavy metals and how they can block protein synthesis.

__**Lab 8**__ Measure HSP70 protein levels using Western Blot.
 * Summary**

//SDS-PAGE// pipette pipette tips Celllytic RT 1.5ml snap cap vials labeled DBB, Protein, 2ND or 3NW depending on ploidy or dry wet 1.5ml screw cap vials pestel microfuge water bath 1x TAE buffer
 * Materials and Methods**

With 5 gill tissue samples and 10 labeled snap cap tubes, I added 500 ul of Celllytic RT and then homogenized my samples using the pestel, which was cleaned or replaced after each use. I then spun my samples and extracted the protein with the pipette into new snap cap tubes. From 4 of these protein stocks 15ul was pipetted into 1.5ml screw cap tube and then 15ul of 2x reducing buffer was added. I centrifuged the sample for 10s then placed it into the water bath where it was kept at >100 degrees C for 5 minutes. While the proteins were being denatured, I helped setup our gel, first assembling the box which holds the gel, then adding 1x TAE buffer till the gel and the electrodes were fully submerged. From my 4 fully denatured protein samples, 3 were selected and pipetted into wells 1-5 on our SDS-Page. We ran a current through the gel for 45 minutes and 150 V. The remaining samples and stock solutions were returned to the -80 C freezer

//Western Blot// oyster DNA 1.5ml snap cap tubes nylon membrane 6x SSC 20x SSC filter paper hot plate beaker centrifuge pipete denaturation buffer UV transilluminator nanopure water plastic dish blocker/dilutent A/B rotary shaker blocking solution 5-MEC antibody TBS-T secondary antibody solution chromogenic substrate ice ice bucket

// WesternBreeze® CHROMOGENIC IMMUNODETECTION // A blocking solution was prepared using 14ml of nanopure water, 4ml of blocker A, and 2ml of blocker. The membrane from our blot was placed into a plastic dish and 10 ml of our blocking solution was poured over. The membrane was then left on the rotary shaker for 30 minutes, after which the excess solution was poured out of the dish. The membrane was rinsed twice with water for 5 minutes on the shaker, then 10 ml of Primary antibody solution was added and incubated for 1 hour. The membrane was washed with 20ml of 1x TBS-T for 5 minutes repeating 4 times, it was then treated with the secondary antibody wash for 30 minutes. After this the secondary antibodies, the membrane was washed with 20ml of water for 2 minutes, then decanted. It was then incubated with 5ml of Chromogenic Substrate until coloring started to appear, then washed again with 20 ml of water for 2 minutes repeating twice. Finally the membrane was placed onto a clean sheet of filter paper to dry.

Unfortunately our antibody bound to a smaller protein and not HSP70.
 * Results**

It is possible that there was no HSP present in these samples and that our antibody found another protein to bind with, what this protein is I am not sure, however there appears to be an equal expression in all of our samples.
 * Conclusion**

It probably wasn't a good idea to do Western Breeze for my project given the complexity of the methods and my limited time frame and availability, but I challenged myself and tried to do something new. I'm not sure what I am supposed to take away from this other than Western Breeze isn't a very dependable method and you should avoid it if you can, and that sometimes discoveries can be made from accidents.
 * Reflection**

The purpose of this was to learn how to measure DNA methylation using a dot blot and how to run a qPCR wash for measuring gene expression.
 * __Lab 7__**
 * Summary**

//Dot Blot//
 * Materials and Methods**

oyster DNA 1.5ml snap cap tubes vacuum manifold nylon membrane 6x SSC 20x SSC filter paper hot plate beaker centrifuge pipete denaturation buffer UV transilluminator nanopure water plastic dish blocker/dilutent A/B rotary shaker blocking solution 5-MEC antibody TBS-T secondary antibody solution chromogenic substrate ice ice bucket labels: DNA, DBB, 1/2/4/8/16

After borrowing a DNA sample, I recorded that the sample was from the ocean acidification oyster project and was treated to high pH water, was triploid, and was not left to dry. I also recorded the DNA concentration and absorbance at 153.1 ng/ul and 1.85 260/280 respectively. First I diluted my solution to 50ng/ul, we wished to have 40 ul total of solution, so using the formula M1V1=M2V2, our desired concentration., volume, and our know concentration, I calculate that I needed to add 26.94 ul of H20 to get 40ul of DNA at a 50ng/ul concentration. I then labeled 5 snap-cap tubes with my initials, "DNA" its respective dilution factor as listed in the lab manual. From here I preceded to create my DNA dilutions adding the respective amounts of DNA stock, H2O, and 20x SSX as indicated by the table in our lab manual. These were placed into a beaker with boiling water, however after the snap cap tubes starting popping we transferred our solutions to labeled screw-cap tubes. After 10 minutes the samples were transferred to an ice bucket to cool, then spun for 5 minutes.

To prepare our Dot Blot, nylon membrane and filter paper was cut to size and soaked in 6x SSC. The manifold was then assembled with the membrane on top of the filter paper. The vacuum was turned on, and 500ul of 6x SSC was added to each well. After boiling and centrifuging, our samples were transferred to their respective wells on the blot. Once all the samples had been filtered through the membrane by the vacuum, the manifold was disassembled and the membrane was placed dots up onto a piece of filter paper soaked pre-soaked in denaturation buffer for 5 minutes, and let to sit for 5 minutes. Next the membranes were put onto dry filter paper and let dry. The dryed blot was then wrapped in plastic wrap and placed DNA side down onto the UV transilluminator for 2 minutes at 120 kJ.

// WesternBreeze® CHROMOGENIC IMMUNODETECTION // A blocking solution was prepared using 14ml of nanopure water, 4ml of blocker A, and 2ml of blocker. The membrane from our blot was placed into a plastic dish and 10 ml of our blocking solution was poured over. The membrane was then left on the rotary shaker for 30 minutes, after which the excess solution was poured out of the dish. The membrane was rinsed twice with water for 5 minutes on the shaker, then 10 ml of Primary antibody solution was added and incubated for 1 hour. The membrane was washed with 20ml of 1x TBS-T for 5 minutes repeating 4 times, it was then treated with the secondary antibody wash for 30 minutes. After this the secondary antibodies, the membrane was washed with 20ml of water for 2 minutes, then decanted. It was then incubated with 5ml of Chromogenic Substrate until coloring started to appear, then washed again with 20 ml of water for 2 minutes repeating twice. Finally the membrane was placed onto a clean sheet of filter paper to dry.

//qPCR// A master mix was prepared for 5 wells wells using 62.5 ul of 2x Immomix, 5ul of Syto-13 dye, 6.25 ul of upstream primer, 6.25ul of downstream primer, and 35ul of ultra pure water. 23ul of master mix was added to 4 wells on my white PCR plate. 2 ul of cDNA was added to the odd wells(1 and 3) and 2 ul of ultra pure water was added to the even wells(2 and 4). The wells were capped and spun for 30seconds. The wells were then placed onto ice after being wiped with a kimwipe, then loaded into the thermocycler and let run.

The dot blot came back showing expression of HSP in my oyster sample, I haven't seen the qPCR results yet.
 * Results**

Based on our measurements the oyster we sampled was under stress before being dissected.
 * Conclusion**

It is interesting to see how different methods of measurement for gene expression, either my measuring RNA, or the protein itself, can produce different results. This is something I will need to take into account when writing my final analysis on the acidification project.
 * Reflection**

My goal for this lab is to begin extracting Protein from my oyster tissue samples and to use these samples in a SDS-Page/Western Plot and measure expression of HSP.
 * __Lab 6__**
 * Summary**

pipette pipette tips Celllytic RT 1.5ml snap cap vials labeled DBB, Protein, 2ND or 3NW depending on ploidy or dry wet 1.5ml screw cap vials pestel microfuge water bath 1x TAE buffer
 * Materials and Methods**

With 10 of my 20 tissue samples and 10 labeled snap cap tubes, I added 500 ul of Celllytic RT and then homogenized my samples using the pestel, which was cleaned or replaced after each use. I then spun my samples and extracted the protein with the pipette into new snap cap tubes. From 4 of these protein stocks 15ul was pipetted into 1.5ml screw cap tube and then 15ul of 2x reducing buffer was added. I centrifuged the sample for 10s then placed it into the water bath where it was kept at >100 degrees C for 5 minutes. While the proteins were being denatured, I helped setup our gel, first assembling the box which holds the gel, then adding 1x TAE buffer till the gel and the electrodes were fully submerged. From my 4 fully denatured protein samples, 3 were selected and pipetted into wells 7-9 on our SDS-Page. We ran a current through the gel for 45 minutes and 150 V. The remaining samples and stock solutions were returned to the -80 C freezer

The lanes containing my samples appeared blank after 45 minutes, indicating either no significant expression of HSP 70, or that the antibodies did not bind to the HSP molecule properly.
 * Results**

Since both samples came from our control treatment, I did not expect any increase in HSP production since the control treatment should be non stressed. I wouldn't be suprised to see some expression in the dry oyster samples once I am able to test them.
 * Conclusions**

It was very exciting to be able to start working independently on our own samples. There was a bit of a bottleneck when I was trying to use the scale and centrifuges, I need to do a better job coordinating with other lab members when using equipment,
 * Reflections**


 * __Lab 5__**

For this lab, we ran our RNA samples from the previous lab through an agarose gel. Then using samples from our oyster dissection during the last lab, I extracted protein from a gill sample for use in our SDS-PAGE gel.
 * Summary**

//RNA Assay//
 * Materials and Methods**

micro-pipette aragose 1x TAE bufer ethidium bromide gel comb UV transilluminator

A gel prepared by our wonderful TA was placed into the gel box and filled with a 1x TAE buffer till all the wells were covered. Combs were removed from the gel and 100bp ladders were loaded with 7ul in each ladder well at the left end of each row. Our RNA samples were then loaded, one containing cDNA, the other with none to act as a control to determine whether our results are accurate. A 100 V current was run through the gel for 1 hour, it is this step that causes our cDNA to separate and tells us to what extents our target genes are being expressed. After 1 hour, the gel was placed onto the UV transilluminator, illuminating each lane, the brighter the tracks, the more target gene expression.

//SDS-PAGE and Western Plot// gel staining tray nanopure water blocking solution rotary shaker Primary Antibody Solution Antibody Wash Secondary Antibody Solution Chromogenic Substrate

After extracting the protein from an oyster gill sample(see Lab 1) taken last week we created a protein stock. From this stock 15ul was pipetted into 1.5ml screw cap tube and then 15ul of 2x reducing buffer was added. I centrifuged the sample for 10s then placed it into the water bath where it was kept at >100 degrees C for 5 minutes. After cooling I centrifuged the sample for 1 minute. Unfortunately there was not enough room for me to run my sample on the assay, however, put I did get to connect and start the assay. After all the samples had been pipetted into the gel, we connected the electrodes and ran a current through the gel at 150 V for 45 minutes. After this time we disconnected the assay, removed the gel, trimmed it, and notched it as a reminder of the correct notification.

Soaking filter paper, membrane, and gel in Tris-Glycine Transfer Buffer for 15 minutes we assembled our blotting "sandwich" with the cathode at the bottom followed by filter paper, gel, membrane, filter paper, and finally the anode. To transfer the blot we ran a current through the sandwich for 30 minutes at 20 V, and rinsed the final product the next morning using a primary antibody wash, a general antibody wash, and a secondary anti-body wash allow for 5 minutes for each wash. After the secondary was, another general antibody was is applied followed by a pure water rinse. Chromogenic substrate is then used to illuminate the secondary antibodies into a bright purple band.


 * Results**

The PCR assay illustrated that there was a significant expression of the HSP70 mRNA (CYS1A1) in our gill tissue sample, and thus, the oyster was stressed at the time prior to sampling. From the Western-Plot, we know as well that their HSP70 protein present as well.

Using these methods, we could now measure the expression of almost any gene or protein that we choose. The hardest part is ahead of us now, as we must replicate the methods that we have been learning over the past few weeks, dozens of times again with the tissue samples from our projects.
 * Conclusion**

This stuff is really, really complicated and takes a really long time. From lectures I though that a PCR assay was easy, something that could be done in a few minutes, NO! It is a very time consuming process requiring concentration and attention. The western blot does seem a bit less complicated, but equally time consuming given how long it takes to wash the samples. We will need to be diligent and very organized if we want to efficiently run all these samples with any major hiccups.
 * Reflection**

__**Lab 4**__

In this lab we created a working stock with our primers and began our first PCR. We also dissected our oysters, taking tissue samples from the gill and mantle for later analysis.
 * Summary**

//PCR// To create our primer stock, we took the dehydrated primers we designed after our last lab and that had been ordered to these specifications, and rehydrated them using 404.1 ul of Tris EDTA buffer~8.5 pH. 5ul of rehydrated primer was then taken and added to a new labeled snap-cap tube for our master mix. To this was added 125ul of 2x GoTaq Green Buffer, and 105ul of nuclease free-water. 5 PCR tubes were labeled with our initials and their respective tissue samples. In each tube we added 48ul of our master mix and 2ul of RNA extracted from earlier labs. 4 of the 5 tubes were centrifuged to pool their contents, and then placed in the thermocycler to begin the polymerase chain reaction.
 * Materials and Methods**

200ul pipette 1.5ml snap cap tube .5 PCR snap cap tube 2x GoTaq Green Master Mix nuclease free water HSP primers thermocycler microfuge ice bucket Tris EDTA buffer

//Oyster Dissection// First we collected the surviving oysters from treatment and organizing them by wet/dry and triploid/diploid. After putting on a pair of safety gloves, we began open the shells for dissections, to do this a metal oyster shucking knife gets inserted into the hinge, then twisted to pry open the shell. The adductor muscle is then severed from the upper shell and then removed. With the internal organs exposed, tweezers and scissors were used to remove 3 gill and mantle samples from each oyster and placed into pre-labeled sample tubes. The samples tubes were labels for wet/dry(W/D), ploidy(Diploid/Triploid), the date, and the sampler's initials. All the samples were then placed into a snap-tube box and put into the -80 C freezer

gloves lab coat oyster knife tweezers scissors 1.5ml snap cap tubes


 * Results**

From our dissection we produced over 50 samples for later use in genetic and protein analysis. The PCR assaywill tell us to what degree HSP70 mRNA production is occurring, and that the primer we designed works.

With our primers, we can now target specific RNA sequences for quantification, that is to say we can now measure what's going on in different tissues at the genetic level. This process will be integral to our group project, as we are measuring the expression of several genese related to stress using the HSP primer we used for this lab. Now that we have completed the experimental portion of our project and collected our samples, we can test these samples for different indicators of stress to determined whether pCO2 levels, ploidy, and/or drying, have a significant effect on these indicators.
 * Conclusion**

I am curious to see how the PCR analysis will turnout; the process has been much more complex than I thought, but we'll see how it goes when we're running through a few dozen oyster samples. Although we had our treatment oysters die early in the experiment, and some confusion during the sampling process, I think we'll be able to turn our some good data from this project. With our design we were able to compare several different factors that could have an effect on stress including pCO2, ploidy, and time out of water to dry. For my part of the project, I would like to look at ACTH levels, or possibly POMCm however I'm not sure if we can measure this directly, that is the protein, or whether I will need to design a new primer.
 * Reflection**


 * __Lab 3__**

For this lab, we reverse transcribed our RNA samples into DNA in preparation for the assay of our oyster samples using primers that we have designed. We also began our ocean acidification experiment to measure the response in Pacific oysters to dramatic changes in pH.
 * Summary**


 * Material and Methods**

//Reverse Transcription// After removing from the ice, we mixed our sample via tube inversion. Next we labeled a new tube with cDNA and Rizky's initials, and transferred 5ul of our RNA, 1ul of oligo dT, and 4ul of nuclease free H2O. This was incubated for 5 minutes at 70 degrees Celsius in a thermocycler. After incubation we placed the sample on ice for minutes then centrifuged and added 5ul of M-MLV 5X buffer, 5ul of dNTPs, 1ul of M-MLV RT, and 4ul of nuclease free H2O. We incubated again at 43 degrees for 60 minutes, then heated to 70 Celsius for 3 minutes in the thermocycler. This was centrifuged and then placed on ice for storage at -20 Celsius.

200uL pipette 0.5mL snap cap tube nuclease free H2O thermocycler centrifuge M-MLV 5X reaction buffer M-MLV reverse transcriptase nuclease free H2O dNTPs

//Ocean Acidification Experiment// For this experiment, 15 triploid/hatchery reared adult oysters and 15 diploid/oyster were placed into mesh bags and put into 2 treatments of sea water:one representing acidified conditions, the other normal sea water/control. The pH was measured and is being measured daily until our next lab session when the oysters will be removed and tissue samples will be taken for a quantitative PCR.

mesh bags trash cans pH probe flex tubing

//Primer Design// We decided to use primers that could measure the expression of heat shock protein since many stressors including changes in sea water acidity, can induce its expression, and because it can be expressed on a relatively short time scale.

Now that we have our RNA made into cDNA, we can finally perform our quantitative PCR and measure the genomic expression of the oyster heat-shock protein, this is the last step in the process before we repeat it with our own samples. This week we will be removing our remaining oysters from treatment and taking tissue samples from various tissues. These will then be tested for the expression of the heat-shock protein using the methods we have learned over the past few weeks.
 * Conclusion**

This lab showed us how to change RNA to DNA for a quantitative PCR. Many of the steps were similar to ones from previous labs; adding reagents, heating/incubating to encourage reactions, centrifuging to separate products, and cooling to prevent denaturing. All of these methods will be necessary when we begin testing our own tissue samples form the acidification experiment. It would be interesting look at how the expression for genes coding for pearlin, a protein associated with calcification of the oyster shell, is affected as well, however, I expect that it would be more pronounced at earlier life stages, and that it would take a longer study to see any changes.
 * Reflection**

October 16, 2011 FISH 411 Steven Roberts

__**Lab 2**__


 * Summary**

To finish isolating RNA from our tissue samples and to find the concentration of our RNA.


 * Material and Methods**

//RNA Extraction and Concentration Quantification// Using our gill sample from lab 1, we removed our sample from ice then brought it back to room temperature(~23 degrees Celsius). We then took it to the fume hood and added 200uL of chloroform. We then vortex our sample, transforming it from clear to white and opaque, and then incubated it for 5 minutes at room temperature. Next, we spun our sample in a microfuge at max speed for 15 minutes to separate the RNA from other cell debris, the RNA, approximately 350 mL, was pipetted into another snap-cap tube and labeled. 500uL of isoproponal was then added and the tube was mixed via inversion, we incubated the tube at room temperature for 10 minutes, then placed it back into the microfuge for 8 minutes. After removing our tube from the microfuge, we extracted the residual supernatunt and then added 1 mL of 75% ethanol to the tube, next we vortex-ed to dislodge our RNA/salt pellet, and placed the tube back into the microfuge for 5mins at 7500g. Next we removed the majority of the ethanol with a pipette, briefly spinning again to collect the excess at the bottom of the tube for removal with a P10 pipette. We allowed the pellet to dry with the cap open at room temperature for 4-5 minutes, next we added 100uL of 0.1%DEPC-H2O and dissolved the pellet using a pipette, and then placed the tube into the hot water bath for 5 minutes to incubate at 55 degrees Celsius. After incubation we removed the sample, briefly vortexed it, and placed it on ice for later concentration quantification.

Taking our sample to the Nanodrop machine we pipetted 2µL of 0.1%DEPC-H20 onto the Nanodrop pedestal, lowered the arm, and calibrated the machine using the "Bland" function within the software. We then pipetted 2 uL of our RNA sample onto the pedestal and lowered the arm. Next we measured the A260/280 ratio at 1.83, the A260/230 ratio at 1.64, and our RNA concentration at 281.2 ng/uL. After wiping the nanodrop with a kimwipe we gave our sample to our wonderful TA Emma for storage at -80 degrees Celsius.

start time(from first incubation):13:36

pipettes-P10 and P1000 200uL of chloroform 1mL of 75% ethanol 1 1.5mL snap-cap tube microfuge 100uL of 0.1%DEPC-H2O hot water bath tube label-DBB, 10/11, oyster/gill, 281.2 ng/uL Nanodrop RNA quantifier


 * Conclusion**

We had a respectable return from our sample in terms of our concentration of RNA. This should be adequate for doing an assay and learning about the gene expression in the gill tissue. I am curious as too whether the methods involved with this whole process vary depending on the tissues one is using.


 * Reflection**

There is one last step for us to learn and that is how to run the actual assay and to find out the actual gene expressions within this tissue. Once we've done this, we can start our project and begin this process on our experiments. The process should vary much given that we used oysters for this lab and that is what we will be using for our ocean acidification experiments as well. I think our next step will be to figure our a sampling regime in terms of what tissues to sample and at what frequency, by sampling multiple tissues, we could see to what degree the genetic expression varies between cell types and treatment. Given the large amount of steps involved with extracting RNA, I would like to know how the process has changed over the years given that it is much more cost effective today that even before, and whether there are any automated systems that can do this as well.