Evan's+Notebook


 * Lab Update**
 * 4 December, 2013**

Last week we were able to take our tissue samples and extract RNA from each using the same protocol as in our preliminary samples. After RNA was extracted all our samples, including the preliminary samples, were converted into cDNA using the protocol established by the FISH 441 lab. Our qPCR results showed no amplification therefore we decided to loosen the parameters and run a conventional PCR. Once cDNA was isolated, we followed the protocol established by Serb et al. (2013). to prepare samples for PCR. We added 3uL of our cDNA from each sample into PCR tubes along with 12.5uL of green taq/buffer/dNTP master mix, 2.5uL MgCl2 (to facilitate annealing), 1uL each of both forward primers and the reverse primer (all taken from Serb et al. 2013), and 5uL of nuclease free H2O. It should be noted that the Serb et al. used a particular ratio of taq, taq buffer, and dNTPs in their experiment however because we used the pre-portioned master mix we could not control these ratios. Sarah was able to run PCR on her samples and then quantify by running the PCR products through electrophoresis, however the results were not what we expected. We were able to see distinct bands, however they settled in the ~150-200 bp range, whereas our gene product had ought to be in the 500-550 bp range. Our initial conclusion was that amplification again failed and what we were seeing were simply primer-dimer products.

We made the decision to run all our cDNA samples to completion despite the seeming failure of our first gel. Today I was able to prepare my cDNA samples for PCR. Steven suggested that because we were running our PCR off of cDNA rather than directly from the DNA it could be feasible that the bands we observed we our desired gene products in a truncated form. I prepared my eight samples for PCR as well as a negative control which included all components of the PCR preparation with the exception of 3uL of nucleotide free H2O substituted for the cDNA. With any hope, if our bands were the result of primer-dimer products it will show in the negative control. If we do not see bands in the negative control then there is a good chance that the bands do, in fact, represent out desired gene product. The PCR will be run on Thursday, 5 December. Our focus now lies on preparing our presentation ad finalizing our papers.


 * Lab Project Update**
 * 18 November, 2013**

I have been slacking in my updates to to Lab Projects so this will be a big update encompassing everything we've worked on up till now. Most of the subsequent updates should be significantly shorter.

I will begin with the question we are looking at researching. Ocean acidification (OA) is a problem growing in severity as well as awareness. As atmospheric carbon emissions continue to increase, the dissolved CO2 in the oceans is concurrently increasing. This increase in pCO2 results in lowered pH and has several physiological consequences for many aquatic organisms. Much of the research being done is simply looking at the extent to which OA can impact physiology. For our project, we are asking what the impact of OA could be on opsin production in pacific sea scallops (C. //rubida//). Interruptions in opsin could severely impact the lifestyle of scallops. We plan to take samples from the gills, mantle, and eye tissue, all of which have been found to contain some degree of opsin. Using these samples we plan to isolate RNA, recover cDNA, and run primer specific qPCR to isolate the opsin gene. We can quantify the amount of opsin gene expression and compare that across conditions.

We collected scallops from the Puget Sound and brought them the to lab aquarium to hold. we took two scallops and subjected them to a light/no light condition so as to test our primers and determine a baseline for opsin production. We placed one scallop in one of two tanks, each with normal pH seawater. One tank was covered by a tarp to prevent light and the other was placed below an incandescent full spectrum bulb. The scallops were allowed to acclimatize to the conditions and then were removed for sampling. he tissue was extracted and placed into sterile tubes. We froze the samples in a -80 degrees C freezer for later processing.

When we were ready to process the samples, we added 500 uL of TriReagent to each sample and using a sterile mortar, pulverized the tissue into solution. 500 uL more TriReagent was added and the sample was frozen for later processing. After coming back to the sample, we brought the solution back to room temperature and then added 200uL of chloroform and the sample was spun in a microcentrifuge at max speed for 15 minutes resulting in a separation of layer. the top most clear layer was carefully removed and transferred to a clean tube. The samples were then spun again at max speed for another 15 minutes to separate out an aqueous phase. This was again removed and transferred to another clean tube. this solution 500uL of isopropyl alcohol was added and the solution allowed to incubate at RT for 10 minutes. After incubation, the sample was spun for 8 minutes to set a small RNA pellet at the bottom of the tube. The supernatant was removed and the pellet washed with 1mL 75% Etol. The ethanol was removed and the pellet then was resuspended in 100uL DECP-H2O.

After isolating the RNA, we began qPCR however an error in procedure resulted in being unable to continue qPCR and so the samples were frozen at -80 degrees C for future processing.

While processing the preliminary data, we set up the condition tanks for the experiment. we used 7.4 pH water for low pH condition and 7.8 pH for control pH. We had a control and low pH tank set up in both light and dark conditions as described above. The scallops were allowed to acclimatize for 3 days.

Our most recent work resulted in extracting tissue samples from all our tests, using two scallops for each condition. These samples were stored in the -80 degree C freezer to be processed this week in lab.


 * Lab 4**
 * 24 October, 2013**

Lab 4 takes the protein sample prepared in lab 3 and runs that protein sample through SDS/PAGE analysis, a process that separates cellular proteins by molecular weight. After the proteins have been separated on a gel, the protein bands are analyzed using western blot.

__Materials and Methods:__

-micropipettes (1-1000 μL) -sterile filter pipette tips (1-1000 μL) -sterile gel loading tips -1.5 mL screw cap tubes -microcentrifuge tube rack -lab coats -safety glasses -gloves -lab pen -timers -heating block with water bath -tube "floatie" (8 tube capacity) -glass container for boiling water that can accommodate "floatie" -protein gel box (SR provided) -SDS/PAGE gels -gel loading tips -trays for staining gels -power supply -platform rocker/shaker -plastic wrap -2X SDS reducing sample buffer -protein ladder marker -gel running buffer -light box -digital camera -Nanopure water -gel staining tray -Blocking Solution -rotary shaker -Primary Antibody Solution -Antibody Wash -Secondary Antibody Solution -Chromogenic Substrate -timers -lab coats -safety goggles -gloves -SDS-PAGE gel -Tris-Glycine transfer buffer -filter paper -nitrocellulose membrane -semi-dry transfer station

To begin, 15uL of the protein sample is added to a clean screw cap centrifuge tube. To that, 15uL of SDS reducing agent is added. this new sample can be centrifuges for 10 seconds to pool liquid at the bottom of the tube, however simply flicking the sides of the tube seemed to work well. The sample is placed in boiling water and allowed to incubate for 5 minutes. the sample is briefly centrifuges to pool liquid and then the entire sample is pipetted into a designated gel well. The gel apparatus is prepared by rinsing the gel with buffer solution and placing the gel into the gel housing. each well should have buffer pipetted into it to wash out any residue. After all the samples are in individual gels, the housing can be plugged in and allow the current to flow along the gel. The current should be stopped before the dye reaches the bottom of the gel. Once the gel has finished, it can be removed from the housing and the plastic casing. After trimming off the wells with a razor, the gel is ready for western blot testing.

Filter paper can be cut to the size of the gel and placed on one side of the semi-dry transfer station. Two membrane can carefully be soaked in Tris-Glycine transfer buffer for 15 minutes. A sandwich is made with filter paper on the outermost layer, then membrane, with the center being the gel. The semi-dry transfer station can be assembled and plugged in and allowed to run for 30 minutes at 20V. The membrane can be removed after the transfer and rinsed with pure H2O. The membrane is then rinsed and incubated with antibody solution for both the primary and secondary antibody. After this, Chromogenic substrate is added to reveal the desired protein band.

__Results:__

Although I was unable to stay for the final results, I was able to see the stained gel which showed distinctive bands of protein. This separation is a very positive result or the purposes of the lab in learning molecular techniques. When I see the final results I will be able to update this section.

__Conclusion:__

Although not all my results are in, I can confidently conclude that the protein isolation worked. The band separation was evident and some chromogenic substrate bands were beginning to appear before I had to leave, thus I expect one of the distinct protein bands I saw on my gel to immunofluoresce.

__ Reflection: __

The techniques used in this lab were helpful to continue establishing a base for molecular la techniques. It was very helpful to follow along with the staining and immunofluorescence processes.


 * Lab 3**
 * 20 October, 2013**

Lab 3 looks to provide an overview of qPCR techniques. cDNA prepared in Lab 2 is processed with a set of primers designed to amplify the DNA sequence in the proper temperature conditions. The DNA is also tagged with a fluorescent marker which can be used to quantify the sample further.

__Materials and Methods:__

-PCR Plates (white); optically clear caps -1.5 ml microfuge tubes (RNAse free) -Nuclease Free water -filter tips -Opticon thermal cycler -kim wipes -2x Immomix Master Mix -SYTO-13 Dye -microfuge tube racks -ice buckets -timers -cDNA samples (student provided) -micropipettes (1-1000uL)  -sterile filter pipette tips (1-1000uL)  -sterile (RNase free) 1.5mL microcentrifuge tubes  -sterile 2 mL screw cap microcentrifuge tubes  -sterile disposable pestles -spectrophotometer -cuvettes for spectrophotometer -microcentrifuge (refrigerated) or in fridge  -gloves -Kim wipes -lab pens  -safety glasses -CelLytic MT Cell Lysis Reagent (with Protease Inhibitor Cocktail added) -Coomassie Protein Assay Reagent -DI water

The first step of this lab is to generate a master mix using 52.5 uL ultrapure water, 2.5 uL each of the primers, and 62.5 uL fluorescent tag mix. In two of the four PCR templates, 1uL of the cDNA sample is added to each of two of the four PCR templates. In each of the other two, 1uL ultrapure water is added as a negative control. The samples are ready for qPCR. The samples are taken to the Opticon thermal cycler.

After the qPCR has begun, preparation for extracting the proteins can begin. 500uL of CellLytic MT is added to the tissue sample. The solution is then homogenized using a disposable pestle. After homogenization, the tube is inverted several times and spun in a microcentrifuge at max speed for 10 minutes. The resulting supernatant is transferred to a new tube labeled 'Protein.' The sample can then be stored on ice.

__Results:__

This lab had even fewer tangible results than the last. However, skills were developed in PCR technique and protein extraction which of them selves is a positive result.

__Conclusion:__

Again, no quantifiable results prevents us from making any conclusions. Because we do not know whether our samples worked, until I see the results I cannot report any conclusion.

__Reflection__ This lab, like the one before, acted as a teaching method to learn more basic molecular methods. This lab walks us through preparing a DNA sample for PCR and began to introduce us to isolation and extraction of proteins. These are both very valuable techniques to have in our repertoire as we further our study of molecular biology.


 * Lab 2**
 * 13 October, 2013**

The purpose of this lab is a continuation from the last week's lab. Using the Pacific oyster mantle tissue prepared in the previous lab, RNA is isolated out of solution using a chloroform solution. Part of this product was then used with a primer/buffer solution to produce a cDNA sample which can then be stored.

__Materials and Methods:__

-micropipettes (1-1000 μL) -sterile filter pipette tips (1-1000 μL) -1.5 mL microcentrifuge tubes -microcentrifuge tube rack -lab coats -safety glasses -gloves -lab pen -timers -ice buckets -phenol/chloroform waste containers (liquid/solid) -vortex -hot water bath -chloroform -RNase free water -chloroform -isopropanol -75% ethanol -0.1% DEPC treated water

Lab begins with removing our sample from the previous week from the deep freeze and allowing to incubate at room temperature for five minutes. After the sample has acclimated at room temperature, 200uL of chloroform is added to the sample. As chloroform is a highly volatile chemical, care should be taken to expose the chloroform to as little open air as possible. The sample can now be vortexed for 30 seconds, resulting an a cloudy solution. After the vortex, the sample is allowed to incubate at room temperature for another 5 minutes. After the five minutes at room temperature the sample can be centrifuged in the refrigerator for 15 minutes on max speed. After the sample is spun, it will segregate into three layers, the clear, uppermost layer is the layer of interest. This layer is to be carefully transferred into a new microcentrifuge tube as to avoid transferring any of the other two layers. The tube containing the lower two layers can be set aside for disposal at the end of lab. 500uL of isopropanol is added to the new microcentrifuge tube and the new sample mixed by inversion until homogeneous. The sample can now sit at room temperature to incubate for 10 minutes. After a 10 minute incubation, the sample can be spun at max speed in a refrigerated microcentrifuge for 8 minutes. A dense pellet will form at the base of the tube. The remaining liquid is carefully removed from the tube and 1mL of 75% EtOH is added to the pellet. Vortexing the pellet/EtOH solution will dislodge the pellet. The sample can then be spun again at 7500g for 5 minutes. After the sample is spun, the EtOH can be carefully removred as to not draw up any of the pellet. To remove as much EtOH as possible, the sample can be spun again for 15 seconds to pool the remaining EtOH at the bottom of the tube where it can be removed with a small volume pipette. The sample can be allowed to air dry for no more than 5 minutes to remove any remaining EtOH. After drying, 100uL 0.1%DEPC-Water is added to the sample and the pellet is resuspended by vortexing. The sample can then be taken to a 55 degree-C hot water bath where it can incubate for 5 minutes. The RNA quantification process would normally follow but this step was skipped in favor of beginning the reverse transcription process. For this process, 5uL of the RNA sample is added to a 0.5 mL centrifuge tube along with 1uL of oligo dT and 4uL of nuclease free water. The solution can then be incubated at 70 degrees-C for 5 minutes. After the sample has incubated, 15uL of a compound buffer solution is added to the tube. This compound buffer contains dNTPs, a reverse transcriptase enzyme, a electrolyte buffer, and nuclease free water. The sample can then be put on ice for storage.

__Results__:

Because the quant step of this lab was skipped over, there are no tangible results to report. However there were technique building results such as the isolation of RNA and beginning to understand the processes involved in primer selection and producing cDNA.

__Conclusion:__

As there were no reportable results, the conclusions of this lab are minimal. I can only make conclusions insofar as they relate to how I preformed the procedure. There was one misstep that I must include in which I deviated from the protocol. During the final step of the lab, I incubated the sample at 70 degrees-C for 5 minutes //after// the addition of the buffer compound. I am unsure how drastically this will effect my results, however it remains necessary to indicate this deviation.

__Reflection:__

Much like the first lab, this lab focused heavily on molecular lab technique building. As such, it served as a useful tool for learning techniques which i have had little to no experience in. I have read about many of the techniques and compounds/products we are using, but to have the hands on experience of actually running the lab is invaluable.


 * Lab 1**
 * 7 October, 2013**

In this lab, RNA and DNA were isolated from oyster gill and mantle tissue. RNA was isolated and stored for future testing while the DNA was isolated and quantified using the Beer Lambert Law to determine concentration.

__Materials and Methods:__

-micropipettes (1-1000uL)  -sterile filter pipette tips (1-1000uL)  -sterile (RNase free) 1.5mL microcentrifuge tubes  -sterile disposable pestles  -vortex  -ice buckets  -gloves  -lab pens <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; line-height: 1.5;"> -safety glasses <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; line-height: 1.5;"> -TriReagent -1.5 mL microfuge tubes -microcentrifuge tube rack -microcentrifuge (room temperature) -razor blades -DNazol -100% ethanol -75% ethanol -0.1% DEPC water -kim wipes -Nanodrop

The first process of the lab was to isolate RNA from a sample of oyster tissue. The sample was puled from mantle tissue of the Pacific Oyster. It is important to note that the sample as kept on ice as often as possible to prevent degradation of the sample. The first step of the procedure is to add 500uL of TriReagent solution to the RNA tissue sample. Using a disposable pestle, the tissue-TriReagent compound was homogenized. The pestle sufficiently homogenized the tissue, however if needed the sample can be vortexed to promote homogenization The tissue was placed back on ice. An additional 500uL of TriReagent was added to the sample and the sample was subsequently vortexed for approximately 15 seconds. The sample can then be stored at -80 degrees C for future quantification. This ends the first process of the lab.

The second part of the lab involves extraction DNA and quantifying it using the Beers Lambert Law to determine concentration and absorbance (determination of purity). In a process similar to the RNA extraction The DNA tissue sample, also taken from mantle tissue of the Pacific Oyster. 500uL of the reagent DNazol is added to the sample and the sample is homogenized using a disposable pestle. The sample was vortexed for approximately 30 seconds to assist homogenization. An additional 500uL of DNazol is added to the sample and the sample is allowed to incubate at room temperature for five minutes. After 5 minutes the sample is spun in a microcentrifuge at 10,000 x g for 10 minutes. The sample was spun and additional 2.5 minutes to promote formation of the cell material pellet. After spinning, the sample is ready for an ethanol wash. The supernatant is transferred to a new labeled sterile microcentrifuge tube. 500 uL of 100% Ethanol is added to the supernatant and the tube inverted 8 times to mix. The sample is then allowed to sit at room temperature for one minute. DNA precipitates out of solution, however the sample did not appear to precipitate in which case the sample can be centrifuged at 10,000 x g for 2-3 minutes forming a pellet of DNA at the bottom of the tube. The ethanol is pipetted out, removing as much ethanol as possible. 1 mL of 75% Ethanol can be added to the DNA sample and the sample inverted 6 times. After incubating at room temperature for 1 minute, the ethanol can be removed from the sample and the wash repeated again with another 1 mL 75% Ethanol. After the second wash, the ethanol is pipetted out, taking care to remove as much ethanol as possible (a 10 uL pipet was used to to extract the maximum amount of ethanol while avoiding disturbing the DNA pellet). After all the ethanol is removed, 300uL of 0.1% DEPC-water is added to the DNA pellet. The sample is then vortexed for 30-45 seconds to break up and suspend the DNA in solution. The DNA is now ready for quantification with the Nanodrop. After the machine is zeroed with dH2O, 2 uL of the DNA solution is pipetted on the machine pedestal and measured through the computer.

__ Results: __

DNA Concentration: 163.9 mg/L Absorbance ratio 260/280 nm: 1.88 Absorbance ratio 260/230 nm: 1.30

__ Conclusion: __

The primary desired result is an 260/280 Absorbance ration near 2 which indicates a pure DNA extraction. A result of 1.88 indicates a 6% deviation from the desired result. This loss of purity could have come form any one of several steps in the process, however most likely is the result of not extracting all the ethanol during the wash process. The logical next step in this process would be to develop a better technique for washing the DNA pellet and/or a more precise method for removing the ethanol from solution. With this, the percent impurity ought to be reduced to negligible amounts.

__Reflection__:

This foundational lab served as an introduction to the molecular lab techniques of DNA and RNA extraction. With these techniques, it is possible to extract DNA or RNA and then quantify both the purity and concentration of the sample. After isolation of the genetic material, any of several other tests can be run to answer various questions about an organism--including transcription rate, (indirectly) protein production, and DNA sequencing. In this lab, we took for granted how the reagents reacted with the genetic material to degrade one and precipitate the other. I would like to look more into the underlying mechanisms of these reagents.