David+B's+Notebook

qPCR methods? data analysis? user:storercg = = =**November 16 2010**=

Last week in lab we calculated the amount of copper needed for 1.3 ppm unfortunately due to a slight error in the calculations we accidently ended up adding 130 ppm of copper while the oysters didn't die immediately which is what I thought might happen. Friday we commenced tissue sampling my station was sampling of the hemolymph from the heart. The sampling went very well. With the exception of 2 oysters that were already dead before we started the experiment and were mistaken for alive. I expect that the rest of our process will start picking up now that we have our samples to analyze. For this process I will be doing mainly qpcr which i believe we will be starting Tuesday. I also took pictures which i will be posting soon.

=November 9 2010=

Today we discussed the setting up of our class projects. The project i am involved with is the oyster stress project. We set up a date for collecting sample organisms. This we did Thursday and Friday we put the oysters in to the experimental set up. So far our timetable will be good. On tuesday we start the experiment and on friday we will have our tissue sample that we can analyze.

Proposal
Salmon are an economic staple for many countries and an environmental necessity for many ecosystems. So understanding how a changing environment in combination with disease and pollution is affecting salmon eggs from hatching till fry stage, their most vulnerable and important stage of development, is of critical environmental and economic importance. This is also important in a hatchery were the goal is to insure that developing eggs are healthy and less prone to disease and crippling development mutations. While there is evidence that temperature difference has major impacts on development (Wedekind, Kung 2010) and that exposure to heavy metals and toxins affect egg fertility and later development (Stekoll, et al 2009). This lead me to the idea of testing combinatios of different stressors on egg development. Since in the natural environment especially one that has been particularly disturbed these eggs would come in contact with a cocktail of different chemicals and diseases. So I am proposing in my experiment to test the weather these combinations of problems are a sum of each other or wether certain ones can combine and become much more deadly than either would have been on its own.
 * Introduction **

With the salmon run coming back to the hatchery fertilized salmon eggs would be rather easy to procure the question is in the number needed. Roughly between 100-200 eggs per set and at least 2 sets for each condition would be appropriate each set would be in its own incubation tray. The conditions would be: control exposure to heavy metals or other contaminants(PCBs) exposure to warmer temperatures(20-22 C) exposure to pathogens exposure to heavy metals or other contaminants, warmer temperatures, and pathogens exposure to heavy metals and other contaminants, and pathogens exposure to warmer temperatures and pathogens and finally exposure to warmer temperatures and to heavy metals or other contaminants The fish embryos changes noted and photographed while dead embryos would be removed and die-offs would be recorded also tissue samples would be taken to analyze for gene expression through taking the RNA reverse transcribing to cDNA and then performing quantitative PCR on it.
 * Materials and Methods ** **﻿**

The first week would be devoted to experiment set up and egg collection. After that it would be a matter of periodic checking for 3 weeks or so with weekly tissue samples and analysis. The final week would be reserved for data analysis and writing of the paper.
 * TImeline **

Stekoll, et al. (2009). Response of the early developmental stages of hatchery reared salmonids to major ions in a simulated mine effluent. Aquaculture, 172-181. Wedekind, C., Kueng, C. (2010). Shift of Spawning Seaon and Effects of Climate Warming on Developmental Stages of Grayling (salmonidae). Conservation Biology, 1418-1423.
 * References ** **﻿**

**November 2 2010 **
Today was an exciting day of lab because we finally got to use the primers we had designed in testing for gene expression. We also finished up our dot blot analysis.
 * Introduction **

Chromogenic Immunodetection/Dot Blot Analysis We started by taking the dried membrane and adding 20 ml of blocking solution and set for 30 min Pour out blocking solution and then rinse twice with 20 ml of pure water for 5 min put the 10 ml of primary antibody solution on the membrane for 1 hour pour off the antibody solution and save wash with 20 ml of TBS-T then add 10 ml of secondary antibody solution for 30 min wash three times with TBS-T for 3 minutes rinse for 2 min with 20 ml of water Finally incubate in 5ml of Chromogenic substrate it then developed and a picture was taken the picture below is the resulting development
 * Method **

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Below is the results from the dot blot analysis [|MeDotBlot_FISH441_F10_LABLED.jpg]

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Quantitative PCR <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">First the primers were resuspended this was done by moving the decimal over in the nM and adding that many ul of pure water then we mixed the master mix enough for 7 so there was a margin of error These are the quantities for 1 mix and then the amount we used along with the final concentrations of each


 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Component || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Volume 1X || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Volume 7X || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Final Con. ||
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Master Mix, 2X (Immunomix) || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">25ul || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">175ul || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">1x ||
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Syto-13 dye (50uM) || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">2ul || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">14ul || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">2uM ||
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">F Primer (10uM) || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">2.5ul || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">17.5ul || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">2.5uM ||
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">R Primer (10uM) || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">2.5ul || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">17.5ul || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">2.5uM ||
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Sterile H2O || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">16ul || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">112ul || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">N/A ||

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">4. Then we added 48 ul of the master mix to each of 6 PCR tubes 5. I added 2 ul of RNA to tubes 1 and 2; 2 ul of cDNA to 3 and 4; and 5 and 6 i added 2 ul of water 6. then i spun the tubes down and handed them over to be run in the PCR Below is a picture of my PCR results


 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Results ** <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">My results were mixed my dot blot analysis came out good you can see a good gradient from the different concentrations. My PCR results were a bit of a problem while my pcr went great a mistake in one of the earlier labs made it hard to tell if the copper oxidase gene was actually being expressed. My error was most likely when pipetting out the RNA when we separated it. This led to DNA contamination and more of a showing that the gene is in the genome and not weather its being expressed.


 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Discussion ** <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">While it was really cool to see that my primers worked it was unfortunate that i couldnt tell about gene expression because of the contamination. Though mabe i will get another chance to try out my primer if we test copper on oysters for our class project. As for the dot blot methylation tests they were interesting especially when contrasting the salmon with acid treatment with then untreated one

I know you had problems with your lab notebook, but could you add the dot blot picture? user:storercg

= = =October 26 2010=


 * __Introduction__**

Today in lab we ran 2 tests we did a gel electrophoresis test on our PCR products from last time and we started a dot blot/chromogenetic immunodetection assay which detects methylation on DNA this we will finish next lab.


 * __Materials and Methods__**


 * Gel Electrophoresis**


 * 1) Add 1X TAE buffer until gel is completely covered
 * 2) Gels were then loaded with our PCR samples 25 ul and the sample ladder 5 ul
 * 3) The gel was then run at 100V for 55min then 150V for 10 min and finally 85V for 20 min
 * 4) Gels were then set in UV light to show where the DNA was and a pic was taken and posted on the message board

Here is a link to the picture of the ladder and my gel runs the first 2 are the tests the last 2 the blanks

http://2.bp.blogspot.com/_MdDM57wDmt8/TM5ZFUc0TyI/AAAAAAAAABI/lDVpyuoqCnc/s1600/Gel2_PCR_FISH441F10-1.jpg


 * Dot Blot/Chromogenetic Immunodetection Assay**

> ** 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 ||
 * 1) First we selected tissue samples mine was of a crab
 * 2) Next we made different dilutions mine went like this
 * = ** Dilution ** ||= ** TARGET **
 * 1) A nylon membrane that was cut to size was soaked in 6X SSC for 10 min
 * 2) Filter paper cut to the same size was soaked in 6X SSC as well
 * 3) the vacuum dot blot assembly was put together so that the filter paper was under the nylon which was under the wells
 * 4) first run each well was filled with 6X SSC
 * 5) we then boiled our DNA for 10 min to denature then put on ice
 * 6) then the entire amount of each sample 200 ul
 * 7) My samples are in D1-5
 * 8) the vacuum was turned on to filter the samples through
 * 9) after this was done the nylon membrane was placed on a piece of filter paper with denaturation buffer for 10 min
 * 10) then for 5 min the nylon was soaked in neutralization buffer
 * 11) the nylon was then dried in the hood this is where we left off and the rest will be completed next lab


 * __Results__**

I was happy with my results on the Gel run since it showed my PCR had gone well with clear differences between my blanks and my tests. As for the dot blot everything seems to have gone well so far but we will see for sure next time.

__**Discussion**__

My results from the gel test were great news since it shows I could correctly run a PCR this is heartening since we will be getting to use our own primers soon and i wouldnt want to screw that up. The dot blot mechanism was interesting mostly cause I've never seen or heard of that particular method before I am interested to see its completion.

= = =**October 19 2010**=


 * __Introduction__**

This week we reverse transcribed our RNA to cDNA so we could practice PCR techniques in preparation for next lab where we will be using our own primers that we designed. We also made agarose gel to analyze the DNA after we have run the PCR. Good user:storercg What gene were your primers for?user:storercg


 * __Materials and Methods__**


 * **PCR and Reverse Transcription**
 * Thaw RNA sample and add 5 ul to a tube with 1 ul of oligo, and 4 ul of nuclease free water
 * Put RNA sample back in the freezer
 * Put mix in the thermocycler for 5 min at 70 C
 * Centrifuge and then add 5 ul of M-MLV 5X, 1 ul of M-MLV RT, 5 ul of dNTPs, and 4 ul of nuclease free water
 * Put back in the thermocycler at 42 C for 60 min with 3 min at the end at 70 C to inactivate it
 * Spin down and put on ice
 * In the Master Mix tube add 250 ul of GoTaq Green Master Mix 2X, 15 ul MnSD (?) forward primer and 15ul of the p450 reverse primer, and finally add 108 ul of nuclease
 * get 4 PCR tubes label 2 test and 2 blank
 * put 48 ul of master mix in each tube
 * in the blanks put 2 ul of nuclease free water
 * in the tests samples put 2 ul of cDNA
 * then put in the thermocycler with this program in place first 95 C for 5 min, then 55 C for 30 seconds, 72 C for 90 seconds and repeat those steps about 40 times after that run 72 C for 3 min
 * after this put on ice for testing next time


 * Since we ran out of GoTag GoTaquser:storercg I shared with someone else.


 * Agarose**


 * 1) Mix 2 g of agarose gel with 150 ml of 1X TAE
 * 2) Heat until agarose is completly disolved
 * 3) when solution cools enough to touch add 12 ul of ethidium bromide
 * 4) pour gel in a mold
 * 5) after it has set store for next time


 * __Results__**

The agarose gel set nicely and we will use it next time to see how our PCR and reverse transcriptions went by running a gel electrophoresis.


 * __Discussion__**

So far I felt this is gone well and that I will be ready for when our primers come in. Though we will see for sure next week how well my PCR turned out. I have enjoyed the chance to design our own primers since i have never done that myself and cant wait to test mine.

=**October 12 2010**=


 * __Introduction__**

This week we performed 2 procedures. We continued the RNA extraction from last lab and we performed further analysis of the protein we extracted last lab by running a SDS-PAGE. My sample was the Pacific Oyster //C. gigas// Treated with Cu treatment C.


 * __Materials and Methods__**


 * SDS-PAGE**


 * 1) <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Thaw protein solution in snap cap that we prepared last time
 * 2) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Add 15<span style="font-family: arial,helvetica,sans-serif;">µ L of protein solution to 15<span style="font-family: arial,helvetica,sans-serif;">µ L of 2X Reducing Sample Buffer
 * 3) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Put protein solution back in freezer
 * 4) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Briefly centrifuge
 * 5) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Boil sample for 5 minutes
 * 6) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Put together gel box
 * 7) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Centrifuge for 1 minute
 * 8) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Load entire sample into assigned well
 * 9) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Turn power supply on and run gel for roughly 45 min
 * 10) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Turn off power supply and remove gel
 * 11) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Use Coomassie stain on gel
 * 12) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Put on shaker to set stain
 * 13) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Rinse gel with acetic acid to remove excess stain.
 * 14) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Continually replace acetic acid till gel can be clearly read
 * 15) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Once bands are visible record results


 * RNA extraction part 2**


 * 1) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px; text-indent: -0.25in;">Take RNA gathered last lab and Thaw
 * 2) <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;"> Add 200 <span style="font-family: arial,helvetica,sans-serif;">µ L of chloroform under the fume hood
 * 3) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px; text-indent: -0.25in;">Vortex sample for 30 seconds
 * 4) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px; text-indent: -0.25in;">Keep sample at room temperature for 5 minutes.
 * 5) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px; text-indent: -0.25in;">__**For**__ 15min
 * 6) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;">Gently remove tube from the microfuge without disturbing it
 * 7) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px; text-indent: -0.25in;">Transfer the super
 * 8) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px; text-indent: -0.25in;">Add 500<span style="font-family: arial,helvetica,sans-serif;">µ L isopropanol to the RNA
 * 9) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px; text-indent: -0.25in;">Stir and then store at room temperature for 10 min
 * 10) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px; text-indent: -0.25in;">Spin for 8 min at max speed
 * 11) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px; text-indent: -0.25in;">Without disturbing the RNA pellet remove the supernatant
 * 12) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px; text-indent: -0.25in;">Add 1 mL of EtOH to pellet.
 * 13) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px; text-indent: -0.25in;">Vortex to dislodge the pellet from side
 * 14) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px; text-indent: -0.25in;">For 5 min spin at 7500g
 * 15) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px; text-indent: -0.25in;">Remove as much EtOH as possible and then let dry
 * 16) <span style="margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px;"> Add 100<span style="font-family: arial,helvetica,sans-serif;">µ L of 0.1% DEPC-H 2 O and the re suspend the pellet
 * 17) <span style="font-family: Arial; margin-bottom: 0px; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 0px; padding-left: 3em; padding-right: 0px; padding-top: 0px; text-indent: -0.25in;">Put in a 55 degree water bath for 5 min
 * 18) Check the amount of RNA with the nanospectrometer


 * __Results__**


 * SDS-PAGE**

The gel went really well with everyones results coming out really well clear. My sample seems well since it matches some of the other oyster samples. The link below has a picture of my results. The 1st lane is the size standard and the the 4th is my result.

[|Protein+Gel+1+FISH441+F10.jpg]


 * RNA extraction**

My RNA extraction also seemingly went well my results were very high compared to some of the others I saw with 1692.9 Ng/µL I believe these are good results though these high numbers could be potentially from contamination. True user:storercg What about your absorbance ratios? What could they tell you about contamination user:storercg

Reflection? user:storercg

=Oct 5 2010=


 * __Introduction__**

In Lab we performed two main tests procedures user:storercg on the tissue samples provided us. The first we performed an RNA extraction using Trireagent. The second test we performed was a Protein Extraction in which we used Cellytic solution we then tested the amount of protein gained extracted user:storercg with a Bradford Assay. We each had different tissues we got to choose from mine is part of the gills of a pacific oyster (//C. gigas)// and was treated with Cu treatment C. Finally we were to prepare for next time by selecting 3 different genes and finding their coding sequence. The purpose of this lab was to give us experience doing basic genetic procedure that would be used in any genetic study.


 * __Materials and Methods__**

RNA Extraction


 * 1) Got my sample Pacific Oyster Cu treatment C
 * 2) prepared a labeled tube with initials and date DDB 10/5/10
 * 3) I then proceded to add 500 µl of trireagent to the tissue sample
 * 4) Then I homogenized the sample with a provided pestle
 * 5) I vortexed for 15 seconds and turned it in to be stored at -80 degrees C

Protein Extraction and Analysis


 * 1) ** Got sample Pacific Oyster Cu treatment C and noted recorded weight 13 mg **
 * 2) ** Added 500 **µl of CellLytic solution
 * 3) once again used a blue pestle to homogenize the solution
 * 4) inverted the tube a couple of times and the spun it in a refrigerated centrifuge for 10 min
 * 5) I then transfered the supernatant to a new tube that I labelled Protein DDB 10/5/10
 * 6) then we ran Bradford Analysis
 * 7) 2 tubes were used in the first the protein sample was diluted in half by adding 15 µl of protein to 15 µl of water this was labelled Test. In the second 30 µl of water was addedthis was labelled blank>
 * 8) then 1.5 ml of Bradford reagent was added to each tube and samples were left at room temperature for 10 min
 * 9) The sample was then analyzed twice in the spectrophotometer that was set to 595 nm after zeroing it with the blank
 * 10) an average of the 2 reading was taken and this was used to calculate the final amount of protein (0.117)
 * 11) Leftover protein was turned in to store at -80 degrees C


 * __Calculations__**

My calculations were accomplished by plugging our Bradford assay results into the equation we got from the standard curve provided. The equation Y=mx+b with b=0 it could be changed to Y=mx with m provided as 1013.9 x is where our analysis info goes so Y=1013.9(0.117)= 118.626 since the the original sample of protein was diluted by half we double the number from this equation 118.626 * 2= **237.252 µg/mL**


 * __Results__**

The RNA extraction will be stored for use next lab. From 13 mg of tissue I was able to extract 237.25 µg/mL


 * __Discussion__**

While the first part of the lab was part of a series we will be continuing the following weeks so the effectiveness of my RNA extraction wont be seen till later. On the protein extraction I felt I did well since my numbers somewhat matched those of people working around me. The main point of this lab was getting us used to common molecular biology techniques. In addition to this it also helped orient us with the lab and procedure. In preparation for next lab I will be finding 3 gene sequences to bring to class.

Do you have any genes of interest? I know you provided this info for the weekly quiz, but could you could please post them here? user:Caroline

=<span style="color: #222222; font-size: 1.8em; line-height: 1.1em; margin-bottom: 0.2em; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 2px;">**281 Candidate genes for larval growth heterosis ( Crassostrea gigas ) Crassostrea gigas cDNA, mRNA sequence** =

code <span style="font-size: 13px; margin-bottom: 0px; margin-top: 0px; overflow-x: visible; overflow-y: visible; white-space: pre-wrap; width: 50em; word-wrap: break-word; zoom: 1;">**IDENTIFIERS**

EST name:      281 GenBank Acc:   EX151622 GenBank gi:    291621473 code
 * dbEST Id:**      **49616663**

code <span style="font-size: 13px; margin-bottom: 0px; margin-top: 0px; overflow-x: visible; overflow-y: visible; white-space: pre-wrap; width: 50em; word-wrap: break-word; zoom: 1;">GATCAACTGCGGAGTTTGCCAACGTCTCTATGCTATGTGCATCAACTGCTGCATGACGCC ATGCTTCGAATCTTGTGGTGGAATCTTCCATCATTTTAAGAAATGAACATAGCAACTGAT TAAACTTTTCTAGTCTTCGTCTGACTTGTTTTTCAAACTTTATATGTGATTCATTTTTCA CATTGCATCTGTGATAAATAATATTTATATTTCATGTCCAAAGTTTCGGATTTTTCGCCG ATGTGTACATGTTTTTATGTAAGAGAGAAATAAAGTTCCTTTTTATTTAAAGAC code

[|281 Candidate genes for larval growth heterosis (Crassostrea gigas) Crassostrea gigas cDNA, mRNA sequence]

Primer


 * 1 || ATGC ||
 * 51 ||  ||
 * 101 ||  ||
 * 151 ||  ||
 * 201 ||  ||

TTCGAATCTTGTGGTGGAATC ACATGTACACATCGGCGAAA
 * Left Primer 1: ||
 * Sequence: ||||||||||  ||
 * Start: 5 || Length: 21 bp || Tm: 59.9 °C || GC: 42.9 % || ANY: 6.0 || SELF: 3.0 ||
 * Right Primer 1: ||
 * Sequence: ||||||||||  ||
 * Start: 192 || Length: 20 bp || Tm: 60.0 °C || GC: 45.0 % || ANY: 6.0 || SELF: 1.0 ||
 * Product Size: 188 bp || Pair Any: 4.0 || Pair End: 0.0 ||
 * Product Size: 188 bp || Pair Any: 4.0 || Pair End: 0.0 ||
 * Product Size: 188 bp || Pair Any: 4.0 || Pair End: 0.0 ||

=<span style="color: #222222; font-size: 1.8em; line-height: 1.1em; margin-bottom: 0.2em; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 2px;">** Crassostrea gigas multicopper oxidase mRNA, complete cds** =

= =


 * Pair 1: ||  ||   ||   ||   ||   ||
 * Left Primer 1: ||
 * Sequence: ||||||||||  ||
 * Start: 385 || Length: 20 bp || Tm: 60.0 °C || GC: 40.0 % || ANY: 5.0 || SELF: 2.0 ||
 * Right Primer 1: ||
 * Sequence: ||||||||||  ||
 * Start: 586 || Length: 20 bp || Tm: 60.0 °C || GC: 45.0 % || ANY: 6.0 || SELF: 0.0 ||
 * Product Size: 202 bp || Pair Any: 4.0 || Pair End: 2.0 ||
 * Product Size: 202 bp || Pair Any: 4.0 || Pair End: 2.0 ||
 * Product Size: 202 bp || Pair Any: 4.0 || Pair End: 2.0 ||

Forward:tttgtgacgcaatgtccaat Reverse:cccaattgtgattccattcc

=<span style="color: #222222; font-size: 1.8em; line-height: 1.1em; margin-bottom: 0.2em; margin-left: 0px; margin-right: 0px; margin-top: 0.5em; padding-bottom: 2px;">** Crassostrea gigas HSP70 mRNA for 70kDa heat shock protein, complete cds** =

[|Crassostrea gigas HSP70 mRNA for 70kDa heat shock protein, complete cds] <span style="font-size: 1.1667em; margin-bottom: 0.1425em; margin-left: 0px; margin-right: 0px; margin-top: 0.1425em;">2,278 bp linear mRNA

AB122063.1 GI:46359613

Primer


 * Left Primer 4: ||
 * Sequence: ||||||||||  ||
 * Start: 564 || Length: 20 bp || Tm: 60.1 °C || GC: 50.0 % || ANY: 3.0 || SELF: 0.0 ||
 * Right Primer 4: ||
 * Sequence: ||||||||||  ||
 * Start: 767 || Length: 20 bp || Tm: 60.0 °C || GC: 50.0 % || ANY: 4.0 || SELF: 2.0 ||
 * Product Size: 204 bp || Pair Any: 5.0 || Pair End: 2.0 ||
 * Product Size: 204 bp || Pair Any: 5.0 || Pair End: 2.0 ||
 * Product Size: 204 bp || Pair Any: 5.0 || Pair End: 2.0 ||

tcaccgttcctgcctatttc tggtaaggatggacacgtca

code format="genbank" <span style="font-size: 13px; margin-bottom: 0px; margin-top: 0px; overflow-x: visible; overflow-y: visible; white-space: pre-wrap; width: 50em; word-wrap: break-word; zoom: 1;">caagtttgaa acttcgaaac tttgagtcaa ctagactacg attcagtaac agtacataca 61 ataatttgtt ttatctacaa ttgaaaaatt tattcaatcg ctactttcac tttagtgttt 121 acaaacaagc atggcaagta aagctccagc catcggcatc gacctgggaa cgacgttttc 181 ctgtgtcggt gtatttcaac atggaaaagt ggaaatcatc gccaacgacc aaggaaacag 241 aacgacgccc agctacgtcg ccttcacaga cacagaaaga ctgataggag atgcggctaa 301 aaaccaggta gccatgaacg ccaacaatac aatctttgac gccaagaggc tgataggccg 361 caagttcaac gacgacagtg tacagtccga catgaaacat tggccgttca cggtgatcaa 421 tgatggagga aagcccaagc tagaagtgga gttcaagaac gagaaaaaga gatttacccc 481 cgaagaaatc agctcaatgg tgctgaccaa aatgaaggag acagcagaag cctacttggg 541 acaaactgtc cgagacgcag tcgtcaccgt tcctgcctat ttcaacaacg cccagagaga 601 ggccactaaa gacgccggag tgatagccgg tctcaatgtt ctcaggatag taaacgaacc 661 cacagctgct gctctggctt atggtctgga taagaacatt tccggtgaga aaaacgttct 721 catctttgac ctcggaggtg gtacatttga cgtgtccatc cttaccattg atgagggttc 781 catatttgag gttcgctcca ctgcagggga tactcacctg ggaggtgaag atttcgacaa 841 cagaatggtc aatcactttg tgcaggaatt caagcgcaag tacaacaaag acatttcaaa 901 aaataaccgt tcactgagac gactcaggac ggcttgcgag cgtgccaaga gaacactgtc 961 cagcagttct gaggccaaca ttgagatcga ctcgctgttt gagggaatgg acttctacag 1021 caaaatcaca cgggctagat ttgaagaaat gtgtgcagat ttgttccgtg gaaccttaga 1081 acctgtagag aaagccctga gggacgccaa aatggacaaa tctaaaattc acgaggtggt 1141 tttggttgga gggtcgacaa gaattccaaa gatccagaaa atgctgcaag acttcatggg 1201 cggaaaagaa ctgaacaagt cgatcaaccc agacgaagct gtggcctatg gtgccgctgt 1261 ccaagcggct attttgaagg gagataagag tgatgccatc aaggacgttc tcctggtcga 1321 tgtcactcca ctgtctctag gcattgaaac tgctggaggg gtcatgacaa agatcgttga 1381 acgaaatgcc aagattccta ccaaagcatc acagaccttc accacatact ccgataacca 1441 gcccggtgta tccattcagg tgtttgaagg tgaacgagcc atgacaaaag acaataacaa 1501 actaggtact tttgaactga atggaattcc tcccgcccct cgaggtgtcc cacagattga 1561 cgtggagttt gacattgacg ccaacggtat cctgaacgta tcagccaagg acaagagcac 1621 gggaaagtcc aataaaatca ccatcaccaa cgacaaaggt cgtctgagta aggccgacat 1681 tgagagaatg gtgaacgagg ccgagacata caaagaggaa gacgataaac agcgtcagag 1741 gatagcggcc cgcaatcagt tagaatccta tgtcttcacc gtcaaacagg cagcagagga 1801 cacaggagac aaacttcagt ctgaagacaa agagacaata tccagggtgt gcagtgaaac 1861 agtgtcctgg ctcgacaaca atgccctggc agaggttgac gaatatgaat tcaaactgaa 1921 ggaggttcag aaggtgtgtt cacccatcat ggccaaacta caccagaacg ggtccacggg 1981 aaatccagga ccagctagtt ctagtcaggg tccgactgta gaggagatgg attaagtgtg 2041 aagaattact taatgataat aaaacctcaa attgtattat gaacttagat tggtactttc 2101 tatgaaagat gtattaaatg ttgaatgtag tattaaaatt tgtagttgtt aatttatttg 2161 agagcattat tttttttttt tacagattac gatatttttt tttacttata caattgttgt 2221 tattcaatgt tgataaaata aaatcatttg aaaattatct aaaaaaaaaa aaaaaaaa

code