Lab 9 Summary
Date: November 29, 2011
1. Detail at least 2 reasons why your results turned out the way they did. This should be easy to do if your results are "unexpected", but even expected results can have multiple explanations. Really think about this, the answer "because I messed up in lab" (or any variation thereof) is not acceptable.

• I was not frugal this time using pipette tips (sorry!). Though not cost-effective for the lab, it helped prevent contamination between samples and master mixes.
• I was very organized in keeping my samples labeled, trays marked, and communicating with my group. This helped reduce the contamination.
• - emmatsThese should be biological reasons, not methodological!

2. What are two obstacles that you encountered during your lab work and experimental design? Did these obstacles affect your results? Why?

• Our experiment was cut short due to oysters dying. Unfortunately, all but one oyster died within 24 hours of submersion to high pCO2 water. I was planning using these in my analysis, but ended up altering my proposal anyways so it didn't have too much of an effect on me. I wasn't sure that 24 hours would have allowed me to get the results I was looking for
• I ran out of Master Mix for both elongation factor 1 and Hsp70. It should not have affected my results that much because I created the master mix within the 2 pcr tubes for each gene. This was not a large set-back, but meant the master mixes were not the same in those two wells. Unfornately, I did not record which of these samples was affected by this so I can not compare their results as I should.

3. Explain at least one aspect of your research and its results that have a greater impact outside of your own personal learning experience. What would you tell a non-scientist who challenged the importance of your research?

• - emmatsWhat happened to answers to questions 3, 4, and 5?
  • It didn't save this. When I opened this document just now, google told me an unsaved copy had been found. Everthing in italics was on a draft from sSunday morningHsp70 is a stress-related protein. The stress the oysters I studied were subjected to was 24 hours out of water. This information could be used to help tailor aquaculture to reduce oyster stress and increase quality of their products. It could additionally help conservation efforts; being able to identify stressors of these oysters may help to indicate harmful environments to species at risk.

4. What part of your research and analysis has completely stumped you? Is there anything you can do to find the answer or will it always remain a mystery?
I had expected the Hsp70/EF1 to increase for the oysters that were removed from water for 24 hours. The opposite was seen in the data. Perhaps this is because oysters are adapted to deal with exposure to air environments because of changing tides. Because of this, closing their shell may protect proteins in the oyster, decreasing the need for Hsp70. Further research on my part needs to be made about this in order to suggest further experiments.
5. In about 3 sentences each, summarize 2 papers that you are going to cite in your own paper that give insight into the results that you found.

Lab 8 write-up
Date: November 22, 2011

• Summary
  • Ran qPCR for samples from OA experiments for research project. Samples included triploid and diploid oysters exposed to low pH environments.
• Method
  • qPCR:
    • Same procedures as before for 52 reactions: 4 controls, 24 samples with Hsp70 and 24 samples with Elongation factor 1
    • Some changes were made in labeling
      • cDNA samples used were: 2nD, 3nD, 2nW, 2nD from c.gigas
      • labeling of PCR wells
        • one end had the sample (one sample per PCR strip), the other end had the protein (H= Hsp70, or E= elongation factor 1)
        • rows A, B, C from top= sample replication one
        • rows E, F, G from top= sample replication two
        • rows D, H: either held 1 of 2 controls per protein, or were blank
    • briefly centrifuged Master Mix
    • All Hsp70 strips and EF1 3nW and 2nW PCR strips went on plate 2; EF1 3nD and 2nD were on plate 4
  • Differences
    • Ran out of master mix for the last two wells for each protein
    • Had to make master mix within the individual wells
• Calculations
  • Master Mix per protein
    • 12.5*26=325 uL Master Mix (Immomix)
    • 1*26= 26 uL Syto-13 dye
    • 1.25*26= 32.5 uL upstream primer
    • 1.25*26= 32.5 uL downstream primer
    • 7*26= 182 uL pure water
• Results/Conclusion
  • From preliminary analysis, the controls looked clean because their graph was a flat line. The samples all showed single melt peaks, indicating little contamination or dimmers within the primer. Still working on how best to group the results to graph, but from their further analysis of the graphs can be made. Plate 4 still needs to be analyzed.
• Reflection
  • The purpose of this lab was to take the cDNA from our experiments and run qPCR on them. We used this to measure amplification of the selected protein. I am using this information to examine gene expression of Hsp70 after low pH exposure.

Lab 7 write-up
Date: November 15, 2011

• Summary
  • We created PCR wells to be used in qPCR next week from cDNA samples we had from previous labs, as well as created DNA dilutions to be used in Dot Blot to analyze if DNA methylation occurred.
• Method
  • qPCR
    • created a master mix for each reaction (5= 2 control, 2 sample, 1 for volume)
      • 62.5uL Master Mix (Immomix)
      • 5 uL Syto-13 dye
      • 6.25 uL upstream primer
      • 6.25 uL downstream primer
      • 35uL pure water
    • Pipette 23uL of Master mix into each PCR plate well
    • Thaw and spin down cDNA
    • Add 2uL of pure water to controls, and 2uL of cDNA to each well
      • cDNA was created from 2n, W, high pH oyster sample
      • Labeled my side of PCR strip with MT
        • From MT to the right: Control, Sample, Control, Sample
    • cap and spin down
    • put PCR strips on ice
    • check PCR settings, load plate and start
  • DNA Dilution
    • DNA chosen was OA sample 48: C.gigas mantle at high pH
    • Snap cap busted open during water bath so we used screw caps
    • Transferred liquid by setting pipette to 201uL
    • Created the first dilution of DNA to get 50ng/uL per 40uL volume
      • 17.226à 17.2 uL DNA
      • 22.77 à 22.8 uL water
    • Then made the proper diultions

Dilution	Target amount	uL of H2O	uL of 20x SSC	uL of 50ng/uL DNA sample
1	800ng	124	60	16
2	400ng	132	60	8
3	200ng	136	60	4
4	100ng	138	60	2
5	50ng	139	60	1

• Dot Blotting
  • Changed membrane to fit the manifold (72 wells)
  • Soaked membrane for 10 min in 6X SSC
  • Altered filter paper to fit before wetting with 6X SSC
  • Put membrane on bottom and filter paper on top in manifold
  • Put DNA in hot water bath for 10 min to denature, then place on ice immediately
  • Put 500uL of 6X SSC in each well and turn on vacuum
  • Remove DNA from ice and spin down for 5 min before pipetting all of each dilution sample into separate wells
    • Row 5- from top to bottom à 0.8, 0.4, 0.2, 0.1, 0.05ng/uL
  • During the vacuum process, cut filter paper and soak in denature buffer
  • After vacuum filtration, move membrane only to the denature filter paper for 5 min, then move to separate neutralization soaked filter paper for 10 min
  • Dry membrane on new filter paper
  • Wrap plastic around blot, and place on U transluminator (set at 120kJ) DNA face-down for 2 min
    • Only used one tip for this pipetting
• Western Breeze Chromogenic Immunodetection
  • Create Blocking solution: 14mL denatured water, 4mL Blocker Part A, 2mL Blocker Part B
  • Fill dish with 10mL of Blocking solution , put membrane in and cover
    • Set rotary shaker to 1rev/sec and allow membrane sit on for 30min
  • Pour out the blocking solution
  • Rinse membrane for 5 min with 20 mL of water and pour out (repeat twice)
  • Primary Antibody Solution (dilution 1:5000, 10mLs)
    • 10mL Blocking solution
    • 2uL 5-MeC antibody
  • Add to membrane and allow to incubate for 5 min
  • Prepare qPCR
  • Pour out antibody
  • Use 20mL of TBS-T to wash membrane for 5min (x3)
  • Let membrane sit in 10mL of antibody soln for 30 min, then pour out
  • Use 20mL of TBS-T to wash membrane for 5min, pour out (x3)
  • Wash with 20mL of water for 2 min, then pour out (x2)
  • Let sit in 5mL of Chromogenic substrate (should see color development)
    • Anywhere from 1-60 min
  • Wash with 20mL of water for 2 min, then pour out (x2)
  • Place membrane on dry filter paper and allow to dry
• 
  
• Results/Calculations
  • qPCR
    • 12.5*5=62.5uL Master Mix (Immomix)
    • 1*5= 5 uL Syto-13 dye
    • 1.25*5= 6.25 uL upstream primer
    • 1.25*5= 6.25 uL downstream primer
    • 7*5= 35uL pure water
  • DNA Dilutions
    • Concentration1*Volume1= Concentration2*Volume2
    • Concentration 1= DNA concentration initial
    • Volume1= DNA
    • Concentration 2= Target concentration (50ng/uL)
    • Volume 2= total volume wanted (40uL)
      • 116.1ng/uL * V1= 50*40 à V1= 17.226 ~17.2uL
    • Total volume –DNA volume= water added
      • 40-17.226= 22.77 ~ 22.8uL water
  • Dot Blotting

Dot Blot (column 5): negative for DNA methylation in my sample.

• Conclusion
  • Assuming I am reading the Dot Blot correctly, my results did not turn out as expected. My results were in the 5th row from the left (I believe) and it appears the results were very weak and uniform. I can attribute the uniformity to using the same pipette tip with each sample despite having varying grades of dilution (a dumb mistake that will not be repeated in the future). But I would not attribute that to a faint result.The faint result indicates that there is little methylation in my sample DNA. Perhaps I over diluted- this problem will need to be revisited. We made PCR solutions this week and will be analyzing them next week and hoping for some better results!
• Reflection
  • The purpose of the lab was to gain experience with creating PCR mixtures, calculating DNA dilution and experience with another blotting method- this time Dot Blot. The procedures can be used to evaluate varying concentrations within DNA, and the expression of a specific gene. I find that a lot of what we do in lab melds together, but after reviewing our procedures, I can piece together the tasks that were correlated. In the future I will be more careful to avoid contamination, especially when dealing with varying concentrations of the same DNA.

Lab 6 write-up
Date: November 8, 2011

• Summary
  • We began the independent portion of our research projects. I worked with Len to extract RNA, and make cDNA for PCR.
• Method
  • Followed protocols from entry in Lab 1 for RNA Isolation with the following changes
    • Removed a 0.025-0.05g of tissue from sample from our experiment: C.gigas, 2n/3n, high pH (control environment), wet all from gill tissue
      • 3 samples from diploid (2n), wet, control (high pH)
      • 3 samples from triploid (3n), wet, control (high pH)
      • **the first sample we weighed was 0.049g which we used to eye-ball the rest
    • Labeled tubes with gill, 2n/3n, high pH, W ad our initials
  • Followed protocols from entry in Lab 2 for RNA Extraction with the following changes
    • The heat bath was removed, and instead more time was spent using a pestle
    • Max speed was set at 13.2
    • Aquaphase was transferred to new tubes labeled: 3n/2n, W, MT
    • After incubating for 10 minutes at RT, we spun down the tubes in a cold microfuge
    • In the spin after the EtOH addition, the fridge had been left open so much that the temperature was definitely not at -4C anymore, probably closer to RT (though it wasn’t measured)
  • Followed protocols from entry in Lab 4 for PCR, cDNA and reverse transcriptase with the following changes
    • We made a Master Mix for 13 PCR reactions (12 with samples, one for an extra incase of pippetting error)
    • While creating the MM we kept the cDNA on ice with RNA
    • Pipetted 15uL of MM to each sample, then centrifuged briefly before putting in the thermocycler
    • We set the thermocycler to finish at -4C so that the samples could be removed in the morning
    • ***We kept the RNA we extracted on ice during this whole process instead of returning to the -80C fridge as instructed in the protocol. This may contribute to any problems/errors in RNA Quantification results when we do that
• Results and Calculations
  • Eyeball sample actual weight: 0.049g
  • Master Mix calculations (for 13 rxns)
    • 5uL M-MLV 5x raction buffer x 13 = 65 uL
    • 5uL of dNTPs x 13 = 65uL
    • 1uL of M-MLV RT x13 = 13uL
    • 4uL of nuclease free H2O x13= 52 uL
• Conclusion
  • I hope my results will turn out as I expected, other than RNA Quantification in the future. Leaving the RNA on ice instead of putting it immediately in the -80C freezer might alter our results in a less than ideal way. Next we plan on doing some qPCR and the gene expression in diploids versus triploids.
• Reflection
  • The purpose of the lab was to allow us to be in charge of our own experiment and gain experience in organizing and experimental procedures and working as a team. The RNA we extracted could be used to quantify RNA, and the PCR mixture can be used to measure gene expression of Hsp70. In this lab we realized that lack of experience can hamper the speed of executing a lab procedure. In the future I plan to think about this in time allocations in the future.

Lab 5 write up
Date: November 1, 2011

• Summary
  • Used samples taken from experimental specimen to make protein stock solution. This was used in gel electrophoresis, and SDS page to gain experience with these procedures. An introduction to Western blotting was also given using Hsp70.
  • Agarose gel
    • In a flask mix gel with ratio of 2g agarose: 150mL 1xTAE
    • Place mixture in microwave for about 3 min.
    • Let solution cool, then mi in 12uL EtBr
      • ***carcinogen- wear gloves!
    • Add to tray, and when set wrap gel in plastic for cold storage
  • Gel Electrophoresis with cDNA
    • Add 1x TAE buffer to gel box after adding gel (should cover wells)
    • §Take out the comb from gel
    • Pipette 7 uL of 100bp ladder into the left side pocket
    • The rest of the pockets receive set volts to 100 for about an hour
    • Use a UV transilluminator to see results
  • SDS-PAGE
    • Have hot water ready on plate
    • Bring protein stock to room temperature, and invert to mix
      • Instead we made our protein stock in lab
        • cut from sample a 0.054g piece (used a boat weight to measure)
          • sample removed from tube labeled: 3n, Dry, mantle, YYL
        • labeled new tube : “protein”, 3n, D, mantle, MMT, 11/1
        • added 500uL of CellLytic +pic to sample
        • centrifuged for 10 min
        • remove supernatant and put on ice
    • Put 15uL of both protein and 2X Reducing Sample Buffer into a 1.5mL screw cap
      • *labeled= SDS page, MT, mantle, D, 3n
      • Protein to be returned to the freezer storage
    • Mix sample and centrifuge for 10s
    • Boil for 5 minutes
      • Take this time to wash the gel
    • After boiling, centrifuge for 1 min
    • Load sample into gel using pipette
      • We loaded 29uL of sample into the well
      • We didn’t have a gel micropipette so a normal pipette was used; additionally the whole sample was loaded into the well
    • Put on lid, plug in electrodes, turn on and set power to 150V, and run for 45 min
      • *** This ran for 27 minutes (with no smilies) and it was finished
    • Turn of power, and remove power supply
    • Take off lid and release the tension wedge
    • Remove gel, and crack cassette to expose it
    • Trim the wells before notching a corner
      • We notched the top right corner
  • Western Blot
    • Soak or saturate paper, membrane and gel
    • The blotting sandwich should look as follows:
      • Anode (+)
      • Filter paper (roll over it to remove bubbles)
      • Membrane (use forceps to place on filter paper)
      • Gel
      • Filter paper
      • Cathode (-)
    • Put blot on 20V for 30 min
    • Take out gel and rinse with buffer
      • Stain with Bradford Assay
    • Use 20 mL of pure water to rinse for 5 min x2
    • Membrane goes in plastic box + 10mL Blcokking solution
      • Incubate and cover through the night in a shaker with the settings 1rev/second
    • pour out the liquid
    • rinse with 20mL water for 5 min x2, then pour out
    • put in 10mL of Primary Antibody Solution and incubate, then pour out
    • rinse with 20mL Antibody Solution for 5 min then pour out x3
    • use 10 mL secondary antibody solution to incubate for 30 min, pour out
    • use 20ml antibody wash to rinse membrane for 5 min then pour, x3
    • use 20 mL of pure water to rinse for 2 min, then pour x2
    • in 5 mL chromogenic Substrate, incubate until the appearance of a purple band
      • typically between 1-60min
    • use filter paper to dry the membrane in open air
• Results

• Real protein layout

  
  The above photo shows which well each individuals protein extraction ended up in. My sample was in slot three and can be seen in a photo of the SDS-page final below.
  
  

  Whole pcr gel

  Pcr gel row1

  
  This picture shows both rows of a PCR electrophoresis gel. The fluorescent bands represent the amplified product. My partner and I used Hsp70 which has 200 basepairs for the primer. Our sample showed an amplification at 200 and also a shorter basepair length which suggests contamination. Our sample was in wells 6-9 (counting from the top) of the row on the left. Wells 7 and 9 were prepared as negative controls, but some contamination was apparent in the gel. The right picture is a close up of the left hand row.
  
  

  Sds page 1

  
  The SDS-page: The ladder was placed in the far left lane as evident by the red band in that well. The larger proteins stayed near the top of each lane, while the smaller proteins drifted further down the gel. A higher concentration of proteins is indicated by a darker band in gel lanes. From this SDS page, it appears that wells 1 and 4 had high amounts of larger proteins present in their extraction.
  
  

  Western

  
  This is a picture of our Western blot. Had Hsp70 protein been expressed, we would have seen it visible here. The Western Blot shows no signs of Hsp70.
  • Sample for protein stock: 0.054g
  • Electrophoresis Procedure
    • Our sample had a single band with a light contamination band in the controls. Control more faded than the sample. No primer dimmers appeared to be present
• Conclusion
  • Our results were not what we were expecting. Our electrophoresis showed the presence of contamination in our controls which decreases the validity of the experiment results. There were no primer dimers though which is good. Based on this I would want to re-do the experiment again to reduce the contaminants present in the control.
• Reflection
  • The purpose of the lab was to gain experience using the SDS-page, electrophoresis, and be introduced to the Western Blot so we can use them in our sample analysis for our experiments. Electrophoresis is used to see if the amplification of PCR was successful- by comparing the ladder of a specific bp, you can check if the correct sequence was amplified. SDS-page does a similar thing, by separating the proteins pipetted into the wells by their weight- the smaller ones being closer to the bottom and larger ones staying on top. SDS-page divides the proteins so that a Western Blott can be used to tag them with protein specific antibodies. These could be used to examine the presence or expression of specific proteins in a membrane. It would have been nice to have more time for the Western Blot. The demonstration was thorough but hard to grasp in the little time we had.

Lab 4
Date of lab: October 25, 2011

• Summary
  • We rehydrated the primers for our group experiment, creating stock solutions at 10uM and 100uM. We shucked our oysters used in our experiment, taking samples from the gill and mantle and placing them in the cold storage upstairs for further RNA, DNA and protein analysis. We also prepared a PCR
• Methods
  • Rehydrating primers
    • Spin down primer quickly- to be sure everything is collected in the tip
    • Added TE buffer (pH 8.5) to the primer 100uM stock for both the reverse and forward transcriptase
      • To know how much of the buffer to add to each, you find the nm on the label of the primer and multiply it by ten. This number wll be the uL of buffer to add
      • Keep this at 35C for 10 minutes
      • After incubation, we vortexed and spun down the stock briefly
      • To use for PCR, made 10uM of working stock and put in a 1.5 mL tube
        • Ratio of 90uL nuclease-free water: 10uL of 100nM stock
  • o PCR Procedures
    • Label 1.5mL micro centrifuge tube with your initials (YM) and MM for master mix
    • Multiply the following measurements for each reagent by the number of PCR reactions being performed
      • The number of PCR reactions should be = (the number of samples taken) + 2 controls + 1 extra to use in case of error
        • Controls= cDNA template should be replaced with nuclease-free water to keep equal volume amounts in each PCR tube
        • Be sure to label each 0.5mL PCR tube with your initials, which organism was used (for example, the experimental conditions) or sample name
          • YM, mantle/gill, D/W (dry or wet), 2n/3n (diploid or triploid)
        • We prepared for 5 PCR reactions
      • Combine reagents in labeled PCR tube
        • 2x GoTaq Green buffer- 25uL
          • 5*25= 125 uL
        • 10uM forward and 10mM reverse primer- 1uL each
          • 1*5= 5uL
        • Nuclease-free water – 21 uL
          • 21*5=105uL
          • We used 2xGoTaq buffer instead of 5xGoTaq buffer
      • In each labeled PCR tube, transfer 48 uL of MM via a pipette
      • Pipette 2uL cDNA template
      • Use the vortex to spin down samples, to ensure mixture is at the bottom of the tube
      • Load the PCR tubes into a thermocycler and use the following settings
        • 1 cycle: 95C for 5 min (denaturation)
        • 40 cycles
          • 95C for 30 sec (denaturation)
          • 55C for 30 sec (annealing)
          • 72C for 90 sec (extension)
        • 1 cycle: 72C for 3 min
        • 1 cycle: 4C (Hold)
      • When finished, keep the samples at -20C
  • Sterile Dissection of C.gigas
    • Leave oysters in the water, then remove immediately prior to dissection
      • Part of our experimental set-up involved removing half of our animals from the water 24 hours prior to dissection (to examine stress responses). Because of this, not all our oysters remained in water before dissection
      • Be sure to use protective gloves (we used non-latex gloves in addition)
      • To shuck your oyster, find hinge and insert shucker there
      • From here wedge shucker between the shells, and slide/twist the blade around the circumference of the oyster. The goal is to slice the adductor.
      • Another option is to find the adductor and insert the shucker there, slicing the adductor and twisting to open the oyster shell.
      • Once open, remove the tissue sample- a larger sized sample is preferable so there is more to work with
        • We removed samples from both the mantle and the gill
        • Be sure to use forceps and a scalpel or razor blade to ensure no contaminants from gloves are put on the sample.
        • Clean the tools before each oyster shucking to eliminate cross contamination- bleach then ethanol.
      • Use forceps to place the sample in the appropriate pre-labeled tubes, then place the tubes on dry ice for storage.
      • The remaining parts of the oyster taken to the hood and placed in a waste receptacle there. Be sure to clean out the oyster containers as well.
• Results
  • We ended our experiment with working solutions of the primers, tissue samples for our experiment and PCR tubes.
• Conclusion
  • There weren’t any recorded results. PCR procedures and primer hydration procedures were performed without problem. The oyster shucking was much more difficult than expected. We had some additional problems with oysters getting mixed up when they were getting shucked. Some didn’t get sampled for both the mantle and the gill, while others may have been put in the wrong tube. In the end we had 10 or 11 samples we knew were correct.
• Reflection
  • The purpose of the lab was to get acquainted with procedures relating to PCR, as well as get some sort of field experience (shucking the oysters). PCR procedures could be used in experiments with DNA analysis, especially if researchers wanted specific sequences of DNA to be amplified. Taking tissue samples is a necessary skill for experiments involving any sort of genetic or epigenetic component. Shucking the oysters was quite difficult. Perhaps next time there will be more opportunity to get this skill correct

Lab 3
Date: October 22, 2011
Date of lab: October 18, 2011

Summary

• Made cDNA by adding reverse transcriptase, and primers to our RNA stock sample (made in previous labs from C.gigas) to be used for PCR analysis in the future. We brainstormed and finalized our experimental design surrounding ocean acidifcation and oysters. Lastly, we discussed primer parameters relevant to our experiment.

Methods

• cDNA
  • Remove RNA stock sample from ice
  • Invert the RNA tube multiple times to mix
  • Label a PCR 0.5 mL tube with "cDNA" and initials (YM, cDNA), then put the following in it:
    • 5 microL of stock RNA (we inverted the tube a few times before extracting)
    • 1 microL of Oligo dT primers
    • 4 microL of De-Ionized water
  • Place the tube with the mixture in the thermocycler at 70C for 5 min, then move straight to ice for 1 minute
  • Inverted tube three times before adding the following:
    • **We combined the the following while the sample was in the thermocycler, Then transferred via pipette to the sample tube.
    • 5 microL of dNTPs
    • 5 microL of M-MLV 5X Reaction Buffer
    • 4 microL of deionized water
    • 1 microL of M-MLV reverse transcriptase
  • Vortex the tube to get everything off the wall and into the tip of the tube
  • Thermocycle at 42C for 60 minutes
  • While we were putting together our experiments, our wonderful TA did the following for us
    • Increase the temperature to 70C for 3 minutes to inactivate
    • Put sample tube in centrifuge briefly before storing at -20C or in ice.
• Oyster shucking
  • Included in the procedures were instructions for oyster shucking. We did not perform this in lab this week, so I have not included the directions for this. They will be provided when the oysters are shucked in lab (next lab)
• Primers
  • NCBI and Primer 3 were given to be used to find primers using the following guidelines:
    • Having a base length between 18-30 bases
    • The difference between the melting temperatures of the primers at 2C or less
    • Stay clear of primer hairpins and dimers
    • Stay clear of G/C stretches, especially towards the 3' end
    • At the 3' end of primers, use a G/C clamp
• Experimental design
  • Our initial design could not continue as planned so the following design was put together
  • Our experiment focused on the affects of ocean acidification on triploid and diploid oysters
  • We used two trash cans: one of seawater acquired from the Seattle Aquarium at a pH of 7.24 and another at a pH of 5.24
    • Trash can with pH 7.24
      • Put 12 diploid and 10 triploid oysters in water
    • Trash can with pH 5.24
      • Put 6 diploid and 5 triploid oysters placed in water
      • The trash can at 5.24 was bubbled with CO2 water
    • Every 24 hours the oysters will be checked to see if they're still alive, and the pH will be taken for each trash can and recorded
      • If any oysters are found dead, a tissue sample will be taken
  • After 5 days, we will take the oysters out of the water and to dry for 24 hours before sampling tissue
    • we will use the tissue samples and analyze them for RNA, DNA and specific protein expressions

Results:

• No measurements were taken with this lab. All procedures went smoothly.

Conclusion:

• We executed the lab successfully, but will need to wait for the next lab to see if this weeks lab was successful in preparing cDNA.

Reflection:

• The purpose of the lab was to gain experience in preparing cDNA from stock samples taken. Additionally, we gained experience in creating our own experiment from scratch, which included adjusting for challenges presented along the way. We were also exposed to creating our own primers. The methods used in this lab would be useful to any protein analysis, preparing solutions for PCR, and experiments pertaining to gene expression/regulation. I wish we had talked more about primers; I understand their use, but would have enjoyed more information on them.

Lab 2
Date: October 16, 2011
Date of lab: October 11, 2011

Summary

• Using our homogenized tissue samples of c.gigas gonad tissue (Lab 1), finish our RNA extraction; measuring RNA’s final concentration, as well as the amount of proteins and ethanol, phenol and salts present in the final solution using a Nanodrop spectrophotometer

Methods

• ***pipette tips and snap caps that were in contact with any amount of chloroform were disposed of in waste containers kept in the fume hood
• RNA Extraction Protocol
  • Set temperature of heating block to 55 C and turn on
  • Homogenized tissue sample (from Lab 1- tissue from gonads of c.gigas) should be incubated for 5 minutes at room temperature
    • Our TA had already removed the homogenized tissue sample from the -80C temperature it had been held at from the previous week
  • Add 200 microL of chloroform to the tissue sample beneath the hood
    • Because chloroform vaporizes at a fast rate, the transferring of chloroform via pipette must be done quickly
  • Vortex on high speed for about 30 seconds.
    • The sample should appear to be a milky salmon colored and homogenized
  • Allow sample to incubate at room temperature for 5 min
  • Place sample in a cold refrigerated microfuge at max speed for 15 min
  • Next, carefully remove tube from microfuge without jostly.
    • Disturbing the tube can distrupt the distinct layers: bottom (organic phase), middle (interphase), and top (aqueous phase, from which the RNA will be isolated). Mixing the layers can result in a poor RNA extraction
  • Pipette the aqueous phase from sample into a separate tube, being sure to avoid removing the interphase also
    • We made this transfer from underneath the fume hood
    • We used a pink tube, instead of the blue RNA specific snap caps
      • In the future, we will use the blue RNA snap cap tubes to be safe
  • Dispose of the remaining tube with the organic and inter-phase material
    • We disposed of the organic and inter-phase material in a chloroform liquid disposal beaker under the hood. The tube went into a waste bag under the fume hood as well
  • In the new tube with the extracted aqueous phase, add 500 microL of isopropanol
    • We added this under the fume hood
  • Invert the tube to homogenize the solution. The solution should not appear chucky (our appeared translucent)
  • With the tube with hinge side facing up, place the tube in a refrigerated microfuge for 8 minutes at max speed
  • When finished, there should be a white pellet in the tip of the tube (salts and RNA). We did see this, but if it is not apparent, continue following the procedure anyways.
  • Using a micropipette, remove the supernatant. This will result in only the pellet left in your tube
  • To the pellet, add 1mL of 75% Ethanol, then vortex temporarily so that the pellet is shaken from the wall of the tube
    • We did not vortex our tube at this point, as our pellet was already dislodged
  • Set refrigerated microfuge at 7500g for 5 min, and spin sample
  • Remove the supernatant, taking care to leave the pellet behind
    • Note: you should be removing just under 1000 microL
  • Spin the tube approximately 15 seconds to agglomerate any ethanol left
    • This spin does not need to be made in a refrigerated centrifuge
  • Remove any ethanol left using a small tipped pipette (P10 or P20)
  • Allow sample to air dry (cap open on tube) for no longer than 5 minutes at room temperature
    • We let our sample “dry” for 4 minutes
  • Add 100 microL of 0.1%DEPC-H2O, and mix by pipetting to dissolve pellet
  • Place tube in a incubated water bath held at 55C for 5 minutes. This helps to bring RNA into solution
    • Our sample was already dissolved so we only did this for 1 minute
  • Mix sample by flicking tube a few times, then place on ice
    • This is your stock RNA sample
  • Follow instructions below to Quantitative your RNA sample yield using a Nanodrop spectrophotometer
• RNA Quantification
  • ***remember when not in use, RNA samples should remain on ice
  • Clean the pedestal and arm that touches the pedestal of the Nanodrop with a Kimwipe
  • On the Nanodrop pedestal, place 2 microL of 0.1%DEPC-H2O, then lower the arm of the Nanodrop
  • On the computer screen, click “Blank” to tare the instrument
  • Clean the surface of the pedestal and surface of the arm with a Kimwipe
  • Remove 2 microL of your RNA sample via pipette and relocate onto the Nanodrop pedestal, then lower arm
  • Select “Measure”
  • Record the ratios given and the concentration of the RNA (ng/microL)
    • Our measurements are given below
      • A260/A280: 1.96
      • A260/A230: 1.96
      • RNA concentration: 882.8 ng/microL
  • Clean the pedestal and arm with a Kimwipe in preparation for its next use
  • Label the tube with RNA stock with the following information: your initials, the date, source organism or tissue, “RNA” and the concentration measured in ng/microL
    • Ours was labeled with: “RNA”, YL, 10/11, c.gigas gonad, and 882.8 ng/microL

Results

• A260/A280: 1.96
• A260/A230: 1.83
• RNA concentration: 882.8 ng/microL

Conclusions

• Our extraction of RNA was extremely successful. Our results were what we expected; they fell within the ranges given. The ideal ranges for the ratios of A260/A280 and A260/A230 are 1.8-2.0 and 1.5-2.0 consecutively. Our results fell within the ranges, however were not found to be in the middle of them. We know from the intro to our lab that proteins absorb light at 280nm. For the A260/A280 ratio, our value of 1.96 suggests a higher concentration of RNA in our sample (RNA absorbs light at around 260nm) and a limited concentration of proteins in our sample. In regards to the A260/A230 ratio, our value of 1.83 is closer to the midpoint of the range (1.75). It suggests a higher concentration of RNA in our sample in comparison to ethanol, phenol, or salt with absorbs light at 230nm. From these results alone, it should be expected that our RNA concentration be quite high. This indeed turned out to be the case. Our measured RNA concentration reading was 882.8 ng/microL.
  • Next, we could compare our results with those of our peers in our lab and discuss possible reasons for variability, if there is some.

Reflection

• The purpose of this lab was to introduce us to and gain experience with techniques involved in isolating RNA. In this lab specifically, we were exposed to a Nanodrop spectrophotometer as opposed to the prototype used in Lab 1. The procedures performed in lab were used to culminate a stock sample of RNA from tissue homogenized and prepared in Lab 1. Then using this stock sample, measure the RNA concentration, the presence of ethanol, phenol and salts, and proteins still in solution with our isolated RNA. These methods might be used in a gene expression study, or in a study involving primers. I was unclear why we weren’t using the RNA specific snap caps (the blue tubes) for our experiments. I thought that these blue tubes were meant specifically for RNA so that it was further protected from UV rays. I wish there was more information on how we isolate specific sequences of RNA, or if there is a need to use the cold microfuges for RNA isolation the whole time as opposed to only specific parts.

Lab 1
Date: October 9, 2011

Summary:
We used TriReagent with a gonadal c. gigas tissue sample to extract RNA. Protein was extracted from c. gigas mantel tissue using CellLytic MT. The concentration of protein was measured using a Bradford Assay. Absorbencies measured were compared to a standard concentration curve provided, and concentrations determined after accounting for dilution factors.

Method:

• RNA extraction
  • Measure weight of tissue sample
    • 0.031g of gonad tissue from c.gigas
    • Labeled the light blue RNA specific snap caps with gonad tissue sample inside What did the label say? - emmats
    • Add 500 microliters of TriReagent and homogenize using a pestle
    • Add another 500 micro liters of TriReagent then vortex for about 15 seconds
    • Store at -80C until RNA extraction can be completed
• Protein Analysis and Extraction Part 1
  • Protein extraction protocol
    • Measure the weight of tissue sample
      • 0.008g of mantel tissue from c.gigas
      • Place sample in pink snap cap tube and label with date
      • After placing the tissue sample in the labeled snap cap, add 500 micro liters of CellLytic MT solution
      • Homogenize using a pestle, then inverting the tube a few times
        • We were unable to completely homogenize the mantel tissue sample with the pestle we were given. As a result, our homogenized solution had a small piece of tissue left when placed in the centrifuge. We were careful not to extract the tissue with our supernatant in the following steps
      • Place in a balanced centrifuge at max speed for 10 minutes
      • During this time, label a green snap cap with: “Protein”, your initials, where the sample was from,, and the date
      • Take pink snap cap tube out of centrifuge and transfer the top liquid (supernatant) into green tube
        • We transferred our supernatant using a micropipette
      • When finished, store on ice
    • Protein quantification Protocol
      • Label screw cap with your initials, the date, BA (method used: Bradford Assay) and “Protein”
      • Tube with Sample: Pipette 15 micro liters of protein into screw cap and add 15 micro liters of De-ionized water
        • At this point in our experiment it became clear that the screw caps were not going to be suitable to homogenize the solution in. We transferred our sample and blank from screw caps into snap caps
        • We transferred our sample using a micropipette
      • Homogenize the solution using a micropipette
      • Create a “blank”: add 30 micro liters of DI water to tube
      • Pipette 1.5 mL of the Bradford Assay to both the blank and the sample tube
      • Allow for Bradford reagent to react by inverting the tubes then leave them for 10 minutes at room temperature
      • Place the tube with homogenized sample solution in a cuvette, and the blank into a separate cuvette
      • Using the blank to tare the spectrophotometer
      • Place cuvette with sample into spectrophotometer and measure absorbency. The machine should be set at a wavelength of 595nm.
        • Record these values
        • 1st absorbance with sample cuvette: 0.478
      • Remove sample cuvette, mix with a micropipette and re-measure
        • 2nd absorbance with sample cuvette: 0.480
      • Find the average of the two absorbencies
        • Average: (0.478 + 0.480)/2= 0.479
      • Calculate the protein concentration using the equation given below. Account for dilution of the tissue solution
        • Y=1013.9x, where x = the absorbance found (use average calculated) and y = concentration of protein
          • Y= 1013.9*(0.479)= 485.7 (ug/mL) protein concentration
          • Multiply the y value calculated by 2 to account for dilution factor. This will give you the protein concentration
            • 485.7*2= 971.3 ~971 (ug/mL) protein concentration in original sample
      • When finished, the protein sample should be given to a lab assistant or TA and kept at -20 degree C

Results

• This week’s lab surrounded preparation for future labs where we will finish the experiments we started this week. All of our samples were given to our TA at the end of class to be kept at -80 degree C or -20 degree C.

Conclusion

• We found our absorbencies to be within a reasonable range of the target, according to our TA. We will use our RNA isolated samples to extract RNA from them next week. After extracting RNA from our isolated samples we will quantify it.

Reflection

• The purpose of this experiment was to isolate RNA, and extract protein and analysis it’s concentration. This lab gave us experience with micropipetting, spectrophotometers, and working with different assays. The Bradford assay was used to measure the concentration of proteins by finding the absorbency using a spectrophotometer. The TriReagent was used to extract RNA so that it could be isolated in the following lab. A study involving gene expression of proteins and concentrations of proteins. This could include proteins such as DNA and RNA. Studies examining any proteins and gene expression in the Central Dogma. I wish there had been more information about the specimen, c.gigas.