User:Brian P. Josey/Notebook/2009/11/12

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Setting Up

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:

  • A DNA polymerase to catalyze the synthesis of DNA: We use Taq which is a thermostable DNA polymerase. The authors suggest that 0.5-2.5 units per standard 25-50 μl reaction are used, which is XXX molarity.
  • Two Primers: These are used for the amplification reaction, and they are really important for the reaction. They are carefully selected to give the desired yields, to suppress the amplification of unwanted sequences and to aid in the manipulations of the product. The standard reactions need 0.1-0.5 μM of each of the primers.
  • dNTPs: This stands for deoxynucleoside triphosphates, which are nucleotides with three phosphate groups attached to it. There are four different dNTPs, dATP, dTTP, dCTP and dGTP, which correspond to the four nucleotides and are kept in equal molar amounts. Typically, 200-250 μM of each are required for the standard reaction. Too much will inhibit the reaction, possibly from the sequestering of Mg2+. When you are storing them, you want to keep them at -20°C and spin them down after thawing them out to keep the concentration just right.
  • Divalent Cations: This are ions that have a charge of +2. They have found that Mg+2 works the best. You could potentially use Mn+2, but it will not work nearly as well or as fast as magnesium. Apparently, calcium just plain doesn't work.
  • Buffer: The buffer is used to maintain the pH of the solution, which should be between 8.3 and 8.8 at room temperature. With the standard solution of Tris-Cl the concentration should be about 10 mM. During incubation, the pH of the reaction mixture drops down to ~7.2.
  • Monovalent cations: Monovalent cations are ions that have a charge of +1. The standard of 50 mM KCl will help with amplification, while increasing the concentration will improve the yield of shorter DNA segments.
  • Template DNA: Obviously, it would be impossible to do a PCR without the template DNA. For larger strands of DNA, amplification can be improved by using restriction enzymes that do not cleave within the targeted sequence.

Mg+2 Concentration

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.


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