User:Brian P. Josey/Notebook/2009/11/17

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PCR Preplaning I'm not too sure about the values for DNA but: {{#widget:Google Spreadsheet		key=tTLhOdy28Sc0gSWXsSgemXQ	width=800	height=300 }} I threw this one together this morning. I am still curious about the values for the template DNA, and I will come back to them after talking to Ant. Brian P. Josey 16:50, 17 November 2009 (EST) It turns out that they are fine. PCR This are my notes for what happens during PCR. I have a lot of sources that I cannot keep track of while putting these notes together. For the most part, I am using notes from Molecular Cloning and formatting it off of the Wikipedia page. Steps to a PCR reaction: Denature raising the temperature to 94-98°C forces the hydrogen bonds in the DNA to break, creating two single strands of DNA. Annealing lowering the temperature down to 50-65°C will allow the primers to attach to the single strands of DNA. This is called annealing, when two complementary pieces of DNA are brought together and fuse. If this step happens too quickly, then the strands might not anneal perfectly. Elongation the temperature is raised up to a point where the polymerase will work the most effectively. The polymerase bids to the site with the primers, and extends them along the DNA from the 5' to 3' ends. This starts to create lopsided strands of DNA, where one half of the helix is shorter than the other. A second cycle of denaturing, annealing and extending creates short strands of DNA that contain only what we want. This is then repeated over and over again until many different strands of DNA are produced. Final Elongation this is held for a while to ensure that all of the single strands of DNA are fully extended. After this cycle you can store the DNA at 4°C to do whatever you need with it whenever you need it. From New England BioLabs, a very general procedure for PCR can be found here. The temperatures for the specific steps of the reaction are: Denaturation 95°C for 2-5 minutes 20-30 Cycles of 95°C for 15-30 sec 55-65°C for 15-30 sec 72°C for a minute per 1000 base pairs Final Extension 72°C for 5 minutes Storage 4°C for as long as needed A great animation of what is going on at each step that really helped me get a handle on PCR can be found here. Optimizing PCR With anything there are going to be some problems with how the PCR will play out and any number of things can come in and mess up the reaction. Essentially the major ways that a PCR can go wrong are: Contamination The ugliest word in the English language. We keep our areas clear, the pipettes, tubes, tips and reagents that I use are all going to be different from the ones that Andy and Ant will use. I'm also going to wipe down the lab bench before and after each PCR, just to be safe. Errors in Copying Errors are possible, especially because Taq does not have a way to proof read itself as it goes through the reaction. While the errors are uncommon, if they occur early in the PCR, they can create multiple strands that are incorrect creating problems for us down the line. Magnesium Concentration I have already talked about this in an earlier journal entry. DIY Biology on Steroids When I was searching around for the above information, I stumbled on something that was really cool. It is an article in Scientific American on how to do a PCR at home and you can read the article here. This simplified approach to PCR was developed by Eva Harris from Berkeley. Dr. Harris is a very interesting individual, her basic idea is to take the advances that we take for granted here, like the PCR and make them accessible, and feasible in developing countries. Her work lead to the formation of the Sustainable Sciences Institute which aims to provide the tool necessary to combat diseases at a local level, before the diseases become more problematic than the need to be. To this end, the institute work to provide the necessary tools and funding in areas that normally don't have access to what we take for granted. Her work is very interesting, and I am looking through it to find out more. The specific procedure that Scientific American gives is very simple, and parallels what we do almost step by step. The first thing you do is get cells from inside of you cheek by using a cotton swab and boiling it down. You can then use a centrifuge, made out of blender, to separate all of the junk out of the tubes. For a calibrated pipette the article's author suggests using stir sticks as a cheap alternative. The DNA is then mixed in with some other chemicals, using units of SPU or Smallest Possible Units. The actual reaction takes place in three pots of hot water, all with different temperatures that are monitored with candy thermometers. To go through the cycles you just take the tubes and move them from one pot to the next in the same order that our thermal cycler would. Then you can create a gel electrophoresis, which they highlight in an earlier article, and visualize it using a black light. While it is not that precise or as technical as our approach, it is still PCR and it follow what we do very closely. The best part though is that Harris wrote a book on how to perform PCRs with limited resources. The book is titled A Low-Cost Approach to PCR and goes into much greater detail. Her purpose in creating this book was to supply the know-how to perform PCR with limited resources to counteract different infectious diseases. UNM libraries actually has a copy of it, and better yet, you can read it electronically of the web. Here is the link. I don't know if you have to be on campus to actually read the book, but I am going through it now to see what other ideas Harris came up with. PCR Today I finished up the PCR from above using the given values. The procedure that I did was: Label nine tubes with the date and the concentration of Mg+2 as given in the table, top row. Aliquot the given volumes of water, in μL as given in the table. Aliquot the given volumes of Mg solution as given in the table. The volume of each of the nine tubes is about 71μL at this point. Create a master solution with all the other compounds as given above in the following order: Buffer solution DNA template DNA primers dNTPs Taq Aliquot this master solution in portions of 29 μL into each tube. This gives a tube volume of 100 μL Put into thermal cycler for the night Thanks to Ant for helping me to do this!