User:Brian P. Josey/Notebook/2009/11/12
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So I am starting a project of my own now, and as Anthony put into my notebook yesterday, I am working with the PCR for the DNA project. We have had the problem of the reactions not working, so I am going to look into how Mg+2 ions help or hinder us. Today Anthony and I started setting up my work area and he showed me where everything I need was. I am also reading through the parts of Molecular Cloning by Sambrook and Russell to get a better understanding of what I need to do. What's bellow are my notes from what I did read in lab today. I am going to read the rest of it tonight and update the notes either tomorrow or over the weekend.
Notes for PCR
PCR stands for polymerase chain reaction, and allows for us to build the DNA up. It has seven different components. They are:
Because a large part of my project is determining the optimal concentration of Mg+2 I found the authors notes on the concentration of magnesium ions really interesting. Both the dNTPs and primers bind Mg+2, so the molar concentration of the cation should exceed that of the molar concentration of phosphate groups from both the dNTPs and primers. It is therefore really hard to find out exactly how much magnesium you need. The typical concentration of magnesium is 1.5 mM, but increasing the concentration to 4.5 mM or 6mM has been reported to decrease nonspecific priming in some cases, while increasing in others. The authors note that some companies sell optimizing kits to determine exactly how much is need for a particular combination of primers and templates but it is better to optimize it in the lab. They suggest that you make ten different PCRs with concentration of the Mg+2 varying from 0.5 mM to 5.0 mM in 0.5 mM incriminates, and comparing the yields. After finding the best range, you then use it as an intermediate value, and do ten more varying the concentrations in 0.2 mM incriminates, finding the best yield humanly possible. It is also a good idea to have as little chelating agents or anions that will isolate the cations.