I finished the qPCR on the eye samples, but unfortunately there was no amplification. We decided to continue optimizing the same primers using PCR and following the design of the paper we stole the primers from (http://icb.oxfordjournals.org.offcampus.lib.washington.edu/content/53/1/68.full.pdf+html) but slightly altered the ratio of reagents (due to the green master mix already containing a mixture of taq, taq buffer, and dNTPs). Unfortunately, this new PCR yielded bands on the gel in the 150-200bp range, while the opsin gene should be about 550bp, meaning it was unsuccessful.
PCR:
12.5 uL buffer mixture
1uL primer F
1uL primer R
2.5uL MgCl2
5uL water
3uL DNA
20 November 2013: Lab 8
I extracted RNA from all samples taken last Saturday and made cDNA out of all 8 samples. I plan to finish the qPCR process this week to allow adequate time for data analysis.
Nanodrop Results:
LiExp: 260/280= 1.74, ng/uL= 442.3
NLExp: 260/280= 1.84, ng/uL= 607.0
LiCO: 260/280= 1.72, ng/uL= 405.2
LiExp: 260/280= 1.74, ng/uL= 408.5
NLExp: 260/280= 1.90, ng/uL= 681.0
NLCO: 260/280= 1.72, ng/uL= 446.9
12 November 2013: Lab 7
Our group worked to continue processing our samples and managed to complete DNA extractions on our preliminary set. The experiment was set up the following day by Amanda with the low pH at 7.4 (taken from the water table in the basement of fisheries) and the normal pH at 7.8 (taken from the barrels in the wet lab). Nine scallops were placed in each of the bins containing normal pH (as we had already sampled one from each) and ten were placed in the bins containing low pH. Samples were taken from 2 specimens from each group on Saturday, November 16th from the eyes, mantle, and gill tissue. These samples (totaling 18 for our group) will be processed during the lab next week by each lab member (depending on the tissue).
Nanodrop results (I realized later this was accidentally set to nucleic acid not RNA):
LiA: 260/280=1.94, ng/uL=97.4
LiG: 260/280=1.71, ng/uL=125.9
LiM: 260/280=1.73, ng/uL=95.3
NLM: 260/280=1.66, ng/uL=68.5
NLG: 260/280=1.64, ng/uL=77.6
NLE: 260/280=1.68, ng/uL=37.0
NLA: 260/280=1.76, ng/uL=36.0
LiE: 260/280=1.59, ng/uL=30.3
6 November 2013: Lab 6
This lab was the continuation of out previous preliminary experiment set up. Tissues were extracted from the two specimens from the mantle, eye, adductor muscle, and gills. These were labeled accordingly with the abbreviations LI/NL for light/no light and the first letter of the body part. We received our primers and rehydrated them before creating a diluted working stock of a ratio 1:10 to water. We began the extraction process, stopping after adding the TriReagent to freeze the samples for processing next week. The experiment set up was delayed due to pH probe issues.
Later, we were also unable to set up because the "low pH" was surprisingly still quite high and would not create much of a difference between out experimental and control groups. Set up will commence next lab (hopefully).
29 October 2013: Lab 5
This lab was a little less structured and designed to have us focus on our individual lab projects. I am part of the scallop group and during our lab time, we finalized the methods that we will conduct over the course of our experiment. We were able to acquire over 40 pink scallops (Chlamys rubida) and are planning on using them in four groups: full light & normal pH, full light & low pH, no light& normal pH, no light & low pH. On Tuesday we set up a preliminary test for only two specimens (both in normal pH water) with different light conditions. We will allow them to acclimatize over the next few days and will take tissue samples from the eyes, mantle and gills to test if opsins are present and if our primers work correctly.
22 October 2013: Lab 4
SUMMARY:
This lab was designed to continue our molecular techniques education for protein analysis of oyster tissue.
MATERIALS AND METHODS:
Gel electrophoresis:
1. Place gel inbox and fill with buffer
2. Load qPCR sample with 2ul of loading dye into the well
3. Run the gel at 1000V for 1 hour
Western Blot:
1. Soak filter paper, membrane and gel in Tris-Glycine Buffer for 15 minutes
2. Assemble the blotting sandwich, consisting of an anode, filter paper, membrane, gel, filter papers, and a cathode
3. Transfer the blot for 30 minutes at 20V
4. Wash the membrane before placing in a plastic box with Blocking solution. Incubate overnight on a rotary shaker.
RESULTS:
I obtained bands, plural, in my gel electrophoresis of the qPCR, indicating some form of contamination.
CONCLUSIONS:
My results were as I expected, but not as I had hoped to see. I'm not sure what brought about the contaminants, but it was likely due to operator error. This lab was just meant to teach us the techniques, but if I were doing this for a research project, I would quantify and analyze my results.
REFLECTION:
The purpose of this lab was to teach us protein analysis techniques--quantify the amount of protein present and to view where the DNA was cut, if at all. These procedures may be used to study protein expression under a variety of different conditions. This procedure was quite clear, but I wish I had gotten to do a Western Blot on my own. I feel like I would be much more comfortable with the procedure had I been given the chance to conduct all parts of it.
15 October 2013: Lab 3
SUMMARY:
This lab was meant to further our familiarity with common molecular techniques by teaching us how to conduct qPCR and extract proteins from oyster tissue.
MATERIALS AND METHODS:
qPCR:
1. Prepare master mix:
62.5uL supermix
2.5uL upstream primer
2.5uL downstream primer
52.5 ultra pure water
2. Distribute mixture evenly between 4 wells
3. Add cDNA to two of the well and only water to the other two
Protein Extraction:
1. Homogenize tissue sample and 500uL CellLytic MT solution
2. Place in refrigerated centrifuge for 10 minutes
3. Extract supernatent
*Pacific Oyster tissue sample -- HSP70 gene
RESULTS:
I successfully extracted proteins and did qPCR on my cDNA, but did not get to conduct gel electrophoresis yet.
CONCLUSION:
I did not achieve many results, but did acquire the necessary final products requested during this lab period. Next week we will use the qPCR product to run a gel and view our sample and use the extracted protein to run a western blot.
REFLECTION:
The purpose of this lab, again, was to continue our understanding of modern molecular technique to utilize in our own research experiments for the purposes of this class. The procedures conducted this week aren't used to measure anything but are a middle step to sequence or visualize different molecular products such as protein concentration. These methods may be used to study gene expression in future studies. Again, nothing was very unclear in this lab and it was all quite straight forward.
08 October 2013: Lab 2
SUMMARY:
The purpose of this lab was to continue isolating RNA from whole tissue oyster samples to progress our familiarization with modern molecular techniques and their applications.
MATERIALS AND METHODS:
RNA extraction:
1. Added another 500ul on TriReagent
2. Add 200ul of chloroform and mix vigorously before incubating
3. Remove aqueous phase to a new tube and add 500ul isopropanol
4. Incubate and spin in refrigerated centrifuge before removing supertanent
5. Add 75% ETOH and centrifuge before removing ETOH and removing excess ETOH
6. Resuspend pellet in 100uL of 0.1%DEPC-H2O and incubate
cDNA
1. Add to a tube the following:
5ul of RNA
1ul of oligo dT
4ul of nuclease free water
2. Incubate for 5 minutes and add 14ul of master mix
5ul MMLV reaction buffer
5ul dNTPs
1ul MMLV RT
4ul nuclease free water
RESULTS:
The end result of this weeks lab was to obtain isolated RNA from tissue samples and to use reverse transcriptase to transcribe the RNA into cDNA. I did not "spec" the RNA to measure the concentration I obtained.
CONCLUSION:
Yes, I obtained the expected results as I ended the day with both isolated RNA and cDNA, which can then be used in PCR and then gel electrophoresis.
REFLECTION:
The purpose of this lab was to gained experience with RNA extraction techniques. We didn't use any procedures to measure anything, besides the people that used the nanodrop machine on their RNA samples to measure the concentration of the sample. These methods could be used to sequence RNA samples for comparison . Nothing was unclear in lab and it was all very well informed and easy to complete.
01 October 2013: Lab 1
SUMMARY:
The purpose of this lab was to gain experience in DNA and RNA extractions as well as familiarize us with current molecular techniques and how they are applicable. Tissue samples were taken from both the mantle and the gills of two species of oyster.
MATERIALS AND METHODS:
RNA Oly_M
DNA Pac_M
RNA extraction:
1. Add 500nL TriReagent
2. Homogenize tissue
3. Add 500nL TriReagent
4. Freeze solution overnight
DNA extraction:
1. Add 500nL DNAzol and homogenize mixture
2. Add 50nL DNAzol
3. Spin down and remove supernatent
4. Add 500nL 100% ETOH
5. Remove precipitate and wash with 500nL 75% ETOH
6. Remove ETOH and add 300nL DEPC water. Homogenize mixture.
7. Spec DNA
*Accidentally used smaller tissue sample for RNA extraction, got a new small tissue for DNA extraction
*Only put in half of TriReagent before freezing RNA extration
RESULTS:
131.4 ng/uL
lamba 230
abs. 2.909
260/280 1.90
260/230 0.90
A-260 2.628
A-280 1.380
CONCLUSIONS:
Considering I only isolated DNA today in class, my results were as expected. Upon specing my DNA, it appears to be a little contaminated with ETOH, but with a strong signal and relatively high concentration of DNA. If I were continuing to work with this DNA, we would next create primers and PCR a specific region to be sequenced.
REFLECTION:
These procedures for DNA/RNA extraction were different than protocols I was previously familiar with. They allowed me gain experience in other methods of DNA extraction without using Qiagen kits. The skills I learned in lab today will prove useful in any future molecular work or research as a method to extract DNA/RNA before sequencing. Nothing about lab today appeared to be unclear or lacking in information to me, it was very straight-forward and relatively easy to accomplish.
Interesting gene= CO1, CYB, RAG1: These are all genes that are typically used in population and molecular comparisons, which would be interesting for me to see the structure of oyster colonies.