User:Brian P. Josey/Notebook/2009/11/17
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I'm not too sure about the values for DNA but:
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.
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:
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:
A great animation of what is going on at each step that really helped me get a handle on PCR can be found here.
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:
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.
I finished up the PCR from above using the given values. The procedure that I did was:
Thanks to Ant for helping me to do this!