Josh's+Notebook

Joshua Matlock’s Notebook

On Tuesday, I followed the following formula to prepare qPCR for IL-12.


 * Component || Volume || Final Conc. || New Volume (x70) ||
 * 2x (Immomix) || 25uL || 1x || 1750 ||
 * Syto-13 dye (50uM) || 2uL || 2uM || 140 ||
 * upstream primer, 10uM || 2.5uL || 2.5uM || 175 ||
 * downstream primer, 10uM || 2.5uL || 2.5uM || 175 ||
 * Ultra pure water || 16uL || NA || 1120 ||

On Friday, I extrapolated the data, and posted it on the link https://spreadsheets.google.com/ccc?key=tZBs24Z-6lxd6ZUtwMeDaFg&hl=en&authkey=CNij6u4J#gid=0

The bar graph is posted on the far right, along with the data.

November 29th-December 3rd

On Monday, Reverse transcription was finished on the box samples. The next day, the salmon team was devised into 2 groups, one group hydrated primers and finalized preparations for the remaining samples to be quantified via qPCR. The other group ran a test qPCR on each of the 14 primers. On Wednesday, it was found that only 8 of the primers worked.

Tuesday, November 23rd, 2010. No Updates. The snow, and impending holidays prevented our progress.

Monday, November 15th- Friday, November 19th, 2010

Overview: For the past week, we have started working intensively with our project. What we have done so far goes as follows:

-RNA Quantification -DNA Normalization -RNA Testing -DNAse -Reverse Transcription

Summary:

The project started Monday when members of our group quantified 79 samples of RNA. The results were promising, we verified that we have plenty of RNA to work with. A google spreadsheet was made, and on Tuesday, we were able to normalize the DNA, with a subsequent qPCR test to determine if there was any RNA carryover. Based off of the results of the qPCR, we found 12 carryovers that required DNAse //(samples: 11, 25, 28, 32, 41, 42, 45, 58, 59, 61, 76, 77) //. On Wednesday, we proceeded to treat the 12 samples with DNAse, in addition to preparing the uncontaminated samples with reverse transcription. Unfortunately, due to a minor miscommunication in our lab, all of our samples ended up with an extra micro-liter of buffer. This was not deemed a significant quantity to confound the results of our experiment, so we proceeded with reverse transcription.

Conclusion: Next week we will have our primers in stock, and with our cDNA, we will be ready to perform qPCR. Despite some minor mistakes in lab, things seem to be running smoothly.

Reflection: We need to read the labels more carefully next time to prevent pipetting errors when preparing samples for vital processes. Had this been a different reagent, we could have compromised the timing, and quality of our experiment.

Wednesday, November 10th, 2010.

Overview: Today was simply a brainstorming/organization day. We are laying the foundations to our experiment.

Summary:

We started compiling the details and concepts of our research. Our group will be focusing on two groups of Coho Salmon. Those which are exposed to a cocktail (comprosmising their immune system), and a second group of Coho not exposed to the cocktail. The former was given either LPS, or a Mock condition with a 24 hour or 48 hour treatment for each condition. This latter is exposed to the same set of circumstances without the cocktail.

We also looked at the broad range of genes that we could potentially use for this experiment, particularly with regard to the immune system, and designed primers for each:

-ptgs2b -COX2 -MHCII Antigen -CYP1A -Cortocotropin Releasing Hormone -Liver Fatty Acid Binding Protein -Superoxide Dismutase -TNF Alpha -IL6 -IL12B -SAA -Vitellogennin Receptor -Heat Shock Protein 90 -Xbpd Splice ID

We have not completely polished out our game plan for this project, but we do know that we have to use the following techniques (chronologically):

-Design Primers (In the process of being created) -Quantify RNA -Reverse Transcribe -PCR -qPCR (Treating all samples with DNAse)

Conclusion: Some of the primers may have not been designed on time, but I feel that we have enough to work off of.

Reflection: This project is going to work very tightly with the remaining weeks ahead. I fear that some the time will extend beyond the weekly Tuesday lab, in which case, I would be very pressed for time due to my extensive schedule. I'm sure, however, that if we split the time equally for each member of the group, we can manage our time effectively, finish in time to compile our results, and make sure that each person is getting the right amount of lab hours without overlapping into classes in their schedule.

Intro: Salmon have the tremendous task of swimming upstream to reproduce. Some have been known to travel as far as 900 miles upstream (1). There must be a huge investment of resources for such a difficult task. Identifying the genes that play this role in upstream movement will be the focus of this project

Research Objectives: My research would involve using molecular techniques, and DNA techniques to assess the function, and expression of genes the sockeye salmon utilizes in response to specific stressors.

Approach/Methods: Particularly, I was hoping to assess sockeye salmon gene transcription/function in response to upstream swimming conditions. What genes do these fish use that help them accomplish such a tremendous feat? By looking at qPCR, we could identify the transcription rates of a particular gene physiologically related to the ability for the salmon to swim upstream.

Timeline: Week 6 -would be focused on discussing the logistics of the project, and outlining a plan to carry out over the remaining weeks. Tissue Extraction/Or obtaining an already collected salmon sample, followed by Protein Assays to assess the functioning capacity of the enzymes of interest. Week 7 - We would also be preparing stock protein/RNA solutions, followed by reverse transcriptase Week 8 –We would conduct a PCR, and design a primer for our gene of interest Week 9 –using our Primers, we would perform qPCR on the salmon samples with increasing temperatures. Week 10 –If time is running thin, we can space out these lab techniques to week 10 to finish our project in time.

(1) [|"A Salmon's Life: An Incredible Journey"]. U.S. Bureau of Land Management. Retrieved 2010-11-7.

Wednesday, November 3rd 2010. Lab #5

Overview: Our chromo immunodetection was finished today to follow up on the results of the dot blot procedure. We also did a qPCR test to monitor transcription levels of our primers that we designed earlier during the quarter.

Summary

Chromogenic Immunodetection:

-10 mL of the blocking solution which was prepared during last week was used to incubate the the membrane for 30 minutes on a rotary shaker at 1 revolution/second. -Blocking solution decanted -Membrane was rinsed with 20 mL of water for 5 minutes. Decanted, than the process was repeated. -10 mL of primary antibody solution (1:5000) was prepared as follows: -10 mL blocking solution -2 uL 5-MeC Antibody -Incubated the membrane in 10 mL of primary antibody solution for 1 hour. It was during this time that we set up qPCR. -Decanted primary antibody solution and washed the membrane for 5 minutes with 20 mL of TBS-T. This process was repeated 3 more times. -Membrane was incubated in 10 mL of secondary antibody for 30 minutes, then decanted. -Membrane was washed for 2 minutes (deviation from the regular 5 minutes) with 20 mL of TBS-T. Decanted and repeated 3 more times. -Membrane was washed for with 20 mL of water for 2 minutes, decanted, and repeated twice. -Incubated the membrane in 5 mL of Chromogenic substrate until the color began to develop -Membrane was rinsed with 20 mL of water for 2 minutes, decanted, and repeated twice. -Membrane was dried with a clean piece of filter paper.

qPCR:

We hydrated our primers and prepared as a 10uM concentration with the following procedure:

100uM (x) = 10 uM (100 uL) x = 10 uL

-I added 217 uL of nuclease free water to my forward primer, and 300 uL of nuclease free water to my reverse primer. I then Labeled each tube 100 uM. -Made a 100 uL mix by adding 10 uL of primer to 90 uL of pure water. Labeled the tubes "JKM Lysozyme-F", "JKM Lysozyme-R"


 * **Component** || **Volume** || **Final Conc.** ||
 * Master Mix, 2X (Immomix) || 25µL || 1x ||
 * Syto-13 dye (50uM) || 2µL || 2µM ||
 * upstream primer, 10μM || 2.5μl || 2.5μM ||
 * downstream primer, 10μM || 2.5μl || 2.5μM ||
 * Ultra Pure Water || 16uL || NA ||

Since we needed enough of these reagents for 6 reaction tubes we multiplied the above listed volumes by 7 (One additional so that we had plenty of reagent to distribute). good user:storercg

-We added the 48 uL of the mastermix to each of 6 reaction tubes. -In the first two tubes, I added 2 uL of cDNA (Note that in the very first cDNA, I accidently used JT's sample instead of my own. I realized this mistake shortly after, and used my own sample on the second tube) -In the 3rd, and 4th tubes, I added my RNA samples 2 uL each. -In the final 2 tubes, I used 2 uL of ultra pure water -At this point, I handed my samples to Caroline, who brought the samples to a lab research scientist to run the qPCR.

Results:

media type="custom" key="7516397"user:storercg Conclusion:

The immunoblot produced results opposite from what I expected. The crab shows almost no methylation whatsoever. This is not too different from the CB crab sample, Perhaps crabs, indeed, have small methylation levels. good user:storercg

My qPCR was very messy, no doubt due to the mistake I made earlier when I loaded a cDNA sample that wasn't my own by accident. This is unfortunate, because I cannot tell if this incongruence with the other samples was a result of this mistake, or a different factor when I was making the master mix, or possibly a combination of both factors. I f you look at the y-axis your florescences is very low compared to others. This would indicate to me that there was no amplification. Either there was no gene expression or the primers did not work. user:storercg

Reflection:

I think it might be fun to try carrying out further epigenetic tests with something more epigentically tangible than crabs. The sockeye salmon seems to be a prime candidate in this regard, and I am going to submit my research proposal based on this model organism. user:storercgGood I think it has a great potential to be studied within the remaining weeks ahead, because it is a resourceful organism, and the tissue stockpiles for this organism are abundant throughout campus.

Wednesday, October 24th 2010. Lab #4

//Overview: Today we finished with gel electrophoresis to assess the quality of our PCR from the previous week. Additionally, we started a dot-blot procedure which we will finish next week with chromogenic immunodetection to monitor methylation levels in our respective organisms.//

//Gel Electrophoresis:// //-Placed gel in the gel box, and filled with 1x TAE buffer// //-Removed combs to form wells// //-Each gel box was filled with 7uL of 100 bp ladder in the far left lane// //-Loaded 25uL of my PCR sample into the gel at position 10-13 on the bottom of gel #2// //-The gel was run 3 times at different voltages:// //-100v from 2:20-3:15 pm// //-150v from 3:23-3:32 pm// //-85v from 3:39-3:45 pm// //-We visualized the gel on a UV transilluminator.//

//Dot Blot Procedure://

//-Signed up for 'Crab1' sample.// //-Labled five 1.5 mL snap caps 'Crab1 - JKM 10.24.10' with their respective concentrations//


 * = ** Dilution ** ||= ** TARGET **
 * concentration ** ||= ** ul of H20 ** ||= ** ul of 20X SSC ** ||= ** ul of 50ng/ul **
 * DNA sample ** ||
 * 1 || 0.8 ng/ul || 124 || 60 || 16 ||
 * 2 || 0.4 ng/ul || 132 || 60 || 8 ||
 * 3 || 0.2 ng/ul || 136 || 60 || 4 ||
 * 4 || 0.1 ng/ul || 138 || 60 || 2 ||
 * 5 || 0.05 ng/ul || 139 || 60 || 1 ||

//(Note, the next steps were performed by different students at different times)//

//-The nylon membrane was cut to fit 72 wells.// //-Membrane was soaked in 6x SSC for 10 minutes// //-Filter paper was cut tot he size of the membrane and wet in 6x SSC// //-Manifold was assembled with the membrane lying on top of the filter paper.// //-DNA was denatured after boiling for ten minutes, and immediately chilled in ice.// //-Vaccum was applied to the manifold. 500 ul of 6x SSC was applied to each well.// //-DNA spun in centrifuge for 5 minutes.// //-The entire volume of DNA was applied to 5 wells. (I had the very bottom row, 1-5)// //-Samples filtered through// //-Soaked filter paper (cut to size) in denaturation buffer// //-Transferred membrane to soaked filter paper and waited// 5 10 //minutes.// - Transferred membrane to filter paper soaked in neutralization buffer for 5 min (this was missing from the printed protocol) user:storercg //-Membranes were set out to dry.// //-The dried blot was wrapped in plastic wrap and placed with the sample side down on a UV Transluminator for// //2 minutes at 120 kj. (Immobilizes DNA)//

//Chromogenic Immunodetection//

//20 ml of blocking solution prepared:// //-Ultra filtered water 14 mL// //- Blocker/Dilutent (Part A) 4 mL// //-Blocker/Dilutent (Part B) 2 mL// //-Total Volume 20 mL// //-Membrane was placed in this blocking solution in a covered plastic dish 10 minutes.//

//We did not finish in time to perform the finishing touches, so we will pick this up next week.//

//Results://

//(My sample was the last bottom 4)// //The immunodetection has yet to be published//

//Conclusion://

//Caroline mentioned that if we our first two samples are bright, followed by the two dim controls, our samples// //are of good quality. Judging from this pattern, and the consistency of the first two bright columns, and their difference to// //the control, I believe that this was a successful run. But of course, my untrained eye might be mistaken.// Woohoo first PCR! The bands indicate that there was amplification. Usually we are want only a single band. Multiple bands(indicating multiple products) might be the result of unspecific amplification, so we would try optimizing the temperature profiles or design new primers. user:storercg

//Based off these results, I can subject these samples to further experimentation, which may be the basis for qPCR.//

//Reflection://

//The procedures in the second part of the lab were largely a measurement of epigentics. Epigenetics is the field of biology which pertains to// //the molecular modifications that can affect gene expression, hence, phenotype. However, it does so without directly altering the genetic code.// //One way of measuring this is by monitoring the amount of methylation that occurs in an organism, which is what was entailed in the Dot-Blot/Immunodetection procedure. This was a rather lengthy process, so I will have to read more about this topic to throughly understand it.//

Wednesday, October 17th 2010. Lab #3

//Overiew: Today, we carried out a relatively simple procedure. It involved transcribing RNA into DNA, and subsequently amplifying this DNA. In addition, we learned how to make the agarose gel (which will eventually used to verify that our DNA Amplification was a success)//

//Reverse Transcription://

//-Mixed the RNA sample by inverting a few times.// //-Labeled a .5 ml PCR tube "JKM CDNA 10.17.10"// //-In the same tube, I combined 5 uL of my total RNA, 1 uL of oligo dT, and 4 uL of nuclease free H2O.// //-Incubated the mixture for 5 minutes at 70C on the thermocycler. Transferred to Ice shortly after incubation.// //-Centrifuged.// //-Added the following reagents// //-5 uL of dNTPs// //-1 uL of M-MLV RT// //-4 uL of nucelase free H2O// //-Incubated the mixture for 60 minutes at 42C, then Heat inactivated at 70C for 3 minutes on thermocycler.// //-Spun the sample in a centrifuge.// //-Stored on Ice at 20C.//

//PCR//

//-As soon as the cDNA was prepared, I labeled 4 PCR tubes 1-4.// //-Made a Master mix with the following reagents:// //-250 uL GoTaq Green Master Mix// //- 15 uL Forward Primer. I used Meatllothionein (MNSD)// //- 15 uL Backward Primer// //- 108 uL Nuclease Free H2O// //-Pippeted 48 uL of the Master Mix into 4 of my .5uL PCR tubes.// //-In tubes 1-2 I used 2 uL of cDNA// //-in the remaning 3-4, I sued 2 uL of Nueclease free water.// //-Spun the mix via centrifuging.// //-Caroline Finished PCR with the following protocol://


 * Step || Temperature || Time || Cycles ||
 * Denaturation || 95C || 5 min || 1 ||
 * Denaturation || 95C || 30 sec || 40 ||
 * Annealing || 55C || 30 sec ||^  ||
 * Extension || 72C || 90 sec ||^  ||
 * Final extension || 72C || 3 min || 1 ||
 * Hold || 4C || ∞ ||= 1 ||

//Agarose Gel//

//-Weighed 2 grams of agarose and mized with 150 mL 1x TAE in a 1 L flask// //(The Rest was done by other students, I will include protocol for future reference)// //-Microwaved the solution for 3 minutes// //-Cooled solution and added 12 ml of Ethidium bromide (NOTE: EXTREMELY TOXIC!! WEAR GLOVES!)// //-Mixed by Swirling// //-Gel Combs Added// //-Gel Was put in the fridge, to be used next week.//

//Conclusion//

//---Results of PCR have yet to be published---//

//I used Metallothionein Primers. My guess is that if we followed up with RT-PCR, the sample would have heightened expression of this Metallothionein Gene under heavy-metal conditions because its function is to counteract the toxicity of the copper environment to keep the organism alive.//

//Reflection://

//The purpose of this lab was to familiarize us with the central toolkit of PCR. It is probably one of the most important techniques to know as a scientist because it forms the underpinning for many different areas of further experimentation such as Southern or Northern hybridization.//

//I didn't quite understand the Gel-Electrophoresis as a way to asses the quality of the PCR, though. What I got out of this process was that you need to compare the actual spots, with a control to see if there are any extraneous variables affecting the results, but mechanically, I don't understand why, or how this happens. Hopefully this will become more clear in the next couple weeks.//

Wednesday, October 12th 2010. Lab #2 - RNA extraction and Protein Analysis, Part 2

// Overview: We started this lab by taking Lab #1 a step further. We used our protein samples to separate them on the basis of their molecular weight. Subsequently, we purified our RNA samples, quantified them, and assessed their purity. //

// Our objectives were as follows: // // -Use protein extracted from C Gigas from Lab 1 to mark on SDS-PAGE Gel // // -Complete our RNA Extraction // // -Determine sequences and genes for Primer Design //

// Summary of Procedures: //

// SDS-PAGE Gel //

// -Thawing my protein extract of C. Gigas gill, I took a 1.5 mL screw cap and mixed 15uL of the protein with 15 uL of 2X reducing sample buffer. This buffer has many important applications. It helps break up the tertiary and quaternary structures of the protein, helps distribute an overall negative charge, maintains a safe buffer zone so that the pH doesn't deviate too far, among other things. // // -I mixed the sample by boiling, centrifuged for 1 minute. // // -Caroline showed us how to properly rinse the gel wells. She took the liquid already in the gel box, and rinsed up and down with the pipette. // // -Loaded the entire sample into my designated well (#7) using a gel loading tip. //

Note: From here on out, the SDS-PAGE was set up by Caroline. I will include the protocol for future reference: // -The gel was run for 45 minutes at 150 voltage // // -Just enough Coomasie Stain was added to the gel to mark our samples. // // -The power supply was shut off // // -The lid was removed, in addition to the tension wedge // // -The gel was removed from the box, with the cassette cracked open to access the gel. // // -The wells were trimmed // // -Gel was placed into a container with coomasie stain, with a notch in the corner used to designate the orientation of the gel. // // -A brief 5 minute period of incubation on the shaker. // // -The stain was poured back into its original container. // // -Gel was rinsed briefly with acetic acid, which was subsequently washed down the drain. // // -Roughly 250 mL of 10% acetic acid was added, and incubated. This step extended beyond our lab session time, and the pictures were taken after we had left. //

// __**SDS Page Results (My column was the 7th from the left)**__ //

// RNA Extraction, Session 2 - //

// -After incubating my homogenized C. Gigas for 5 minutes, I added 200 uL of chloroform to my sample behind a fume hood. // // **__NOTE__**: Chloroform must be handled with care! __It is very volatile!__ // // -I vortexed the resulting mixture for 30 seconds, then incubated for 5 minutes. // // -Spun the mix in the centrifuge for 15 minutes at the maximum speed. // // -I very carefully removed as much aqueous phase as I could to a new tube, and added 500 uL of isopropanol, Inverting several times after. // // -Incubated at room temperature for 10 minutes, and spun in centrifuge at maximum speed for 8 minutes. // // -Removed the supernatant, and added 1 mL of 75% EtOH to the pellet. Then I dislodged the pellet by vortexing. // // -Again, I ran the sample through a centrifuge for 5 minutes. // // -Carefully removed the supernatant without removing the pellet. Spun the tube for 15 seconds to extract residual water. // // -Dried pellet for 5 minutes, then mixed in 100 uL of .1% DEPC-H20 by pipetting up and down. I did this until the sample dissolved. // // -Incubated one final time at 55 C for 5 minutes, and stored the stock sample on ice. // // -Took recordings of the RNA purity, and Yield, the values were as follows: //

// A260/280: 1.96 // // A260/30: 1.67 //

// ng/mL = 586.1 //

// Results: //

// My RNA turned out to be pure enough to use as a stock solution. I know that they are pure because the reference values for A260-A280 range between 1.8-2.0. The A260/A230 reference value is 1.5-2.0. Both of my ratios fell within this acceptable range. I believe that this was accomplished by simply taking my time, and not rushing through any of the protocol. Knowing this solution is pure, I can reuse the RNA stock in the future, as needed. //

// As for my Gel Results, I could only spot 3 visible bands on my column (7th from the left), with each band progressively darker down the line. It is interesting to note that 3,4, and 6 also had nearly the same patterns, which suggests to me that we have similar proteins (if not identical) that we are working with. //

// Reflection: //

// I believe that like the previous lab, this lab was designed to familiarize ourselves with the fundamentals of laboratory tools that biologists use on a daily basis. // // The RNA extraction, I hypothesize, is important because it serves as a precursor for identifying particular sequences that will give rise to proteins of interest that are needed under the treatments the sample was exposed to, in addition to it's potential use for reverse transcriptase PCR. //

// I should note that before the SDS-PAGE was run, I had difficulty loading my sample into the well, (having only done this once before). Personally, I believe this was the trickiest part of the lab, knowing where to load the sample in these transparent wells. Hopefully this is a skill that I will acquire through muscle memory if I practice it more in the future. I presume that because the SDS-PAGE sorts different proteins out by molecular weight, it is useful to identify potential proteins, and to assess how different treatments yielded expression of certain mRNA sequences to ultimately give us particular proteins of interest. //

// Unfortunately, I still know very little about the theory behind the molecular laboratory techniques we have been using, and I hope that over time, I will know when will be the appropriate time to use these techniques, and why I need to use them. //

Wednesday, October 5th 2010. Lab #1 – Tissue Extraction

//Overview:// //Today, we began our very first experiment in the environmental physiology lab. My organism of choice was the Crassostrea Gigas //Crassostrea gigas// tip: scientific names should always be in italics user:storercg (henceforth referred to as C. gigas) also known as the pacific oyster. My C.gigas consisted of tissue from the gill, and this sample was given the Cu+ A. treatment.

Our primary objectives of this lab were to: 1.) Extract the RNA from the tissue sample 2.) Extract Protein from the tissue sample 3.) Become Familiarized with primer design

Summary of Procedures:

Part 1 of our lab consisted of extracting RNA from the tissue, and saving the sample for later. Part 2 involved using our second sample to collect homogenized protein, and subsequently measure the concentration using the values fed by the spectrometer. These values were made possible through the use of a Bradford Protein Assay. During Part 3 of the lab, Caroline introduced us to primer design, in addition to searching the NCIB database to locate genes of interest.

Materials and Methods:

RNA Extraction:

- - I began isolating the RNA from my tissue sample by adding 500uL of TriReagent to the cap containing the tissue. Trireagent specifically separates RNA from the cell complex. Good! user:storercg

- - I labeled this blue tube as follows: “C.Gigas Gill Cu A - JKM 10.5.10” - - I obtained a disposable pestle and began homogenizing the sample by smashing the tissue, and vortexing for several minutes. - - Afterwards I added an additional 500uL of Trireagent, and vortexed for about 20 seconds. At this point, I handed my sample to Caroline for future use.

Protein Extraction:

- - I recorded that the weight of the tissue sample was 15 mg - - Labeled the green tube as follows: “C.Gigas Gill Cu A – JKM 10.5.10” - - Added 500 ul of CellLytic MT solution to the tube containing my sample - - Again, I homogenized the tissue with a pestle. However, I inverted the tube instead of vortexing. - - Ran the sample through a centrifuge for roughly ten minutes. - - Labeled a new tube “Protein - C. Gigas Gill Cu A – JKM 10.5.10” - - Transferred the supernatant to the new Protein tube.

Protein Quantification: - - Labeled two additional tubes "Blank", and "Protein BA" - - Transferred 15uL of the protein to the BA tube, and added 15uL of deionized water, mixed by pipetting. - - Added 30 uL of pure deionized wated to the Blank tube - - Added 1.5 mL of Bradford Reagent to both tubes. Inverting each tube afterward, and incubating for ten minutes - - Transferred 1500 uL of each tube to individual cuvettes (Note that this step was incorrect according to protocol, I should have instead only added 1000 uL to each cuvette.) - - Adjusted the absorbency to the blank, then took two readings of the protein sample: .109 and .104. (The second absorbency was recorded after remixing the sample with the pipett, and cleaning the smudges on the side of the cuvette with KimWipe. - - I averaged the two absorbencies and performed the final calculation to obtain a concentration of 215.96 ug/mL. The calculations are provided below:

Y = mX + b b= 0 m= 1013.9 X = .1065

(.1065 x 1013.9 + 0) x 2 = 215.96 ug/mL

(I multiplied by 2 to account for the dilution factor)

Primer Design:

I selected mRNA sequences that would possibly be involved in the response to Cu+ treatment. As such, I picked the gene for Metallothionein gene promotor ( AJ249659) The promotor lies outsides of the "expressed" exonic regions of a gene that are transcribed into RNA so you will not be able to use this nucleotide sequence to design primers of off for measuring gene expression using qPCR user:storercg, Metallothionein exons 1-3 (AJ242657) **Good! I would recommend using this sequence for primer design** since it represents the exonic regions for a gene of interest user:storercg, and Extracellular Superoxide Dismutase (EF694097). While an interesting gene this is the entire gene sequence (not just mRNA or cDNA) which contains both the introns and exons (the expressed regions transcribed into RNA). We will only be able amplify exonic (since it is RNA) regions using qPCR so primers designed off of this sequence might not work because we do no where the exons are. For this class, I would stick to one of the other genes because there is a better chance that the primers will work.user:storercg I believe that transcription rates for these sequences will be higher since additional factors would have needed to be recruited to meet the demands of a heavy Copper environment.

Unfortunately, I have no data to compare this to, so as of yet, I cannot say with complete certainty whether or not my units are around the normal value. If this data is reliable however, it will, of course, be important to take note of for future experiments that require the use of this sample protein. One thing to comment upon in your results is that protein quantification was successful as indicated by the 215 ug/ml of protein quantified using the Bradford assay. user:storercg

Reflection:

I believe that the purpose of this lab is to get us acquainted with basic laboratory protocol so we can build our way up to more complicated tools. The procedures are in this lab were used to quantify the concentrations of protein in our tissue samples. My guess is that this will be pivotal for more detailed analytic techniques, such as identifying gene expression via Western Blotting. It is clear to me that quantifying protein is fundamental for followup experiments, but which specific ones, I am not yet sure. I am hoping to learn more about the importance of protein assays as we progress.