| Contributor: |
The Laboratory of Jasper Rine at the University of California, Berkeley
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| This protocol describes the misincorporation of nucleotides by Taq polymerase in the presence of Mn2+ ions. The dNTP concentrations are adjusted to balance the base substitutions produced. However, due to the properties of Taq polymerase, certain nucleotide mutations rarely occur (e.g., AT to CG and GC to CG). These conditions are reported to give 1.5% mutations for n=10, where n is the actual number of doublings in the PCR reaction (not the number of programmed cycles). However, the actual frequency depends on the base composition of the template DNA. The mutation frequency is proportional to the number of doublings. Therefore, doubling to n=20 should give a frequency of 3%; or reducing the doubling to n=5 gives 0.75%. The mutation frequency is the number of changes per hundred bases in the final product. |
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1. Set up the PCR reactions on ice with the following components:
10 μl of 10X PCR buffer
10 μl of 10X dNTP mix
2.5 μl of 20 μM primer 1 (50 pmol)
2.5 μl of 20 μM primer 2 (50 pmol)
10 μl of 5 mM MnCl2
6.52 μl of 50 mM MgCl2
Add template DNA to a final concentration of 640 pM
Add 8 Units of Taq polymerase
Add ddH2O to a final reaction volume of 100 μl
2. Overlay all the reactions with 100 μl of Mineral Oil.
3. Run the required program of the thermocycler that optimizes the amplification of the desired product.
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| 5 mM MnCl2 |
| 50 mM MgCl2
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| 20 μM Oligonucleotide Primers |
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| dNTP mix (10X) |
| 0.90 mM dCTP
0.56 mM dATP
0.20 mM dGTP
1.40 mM dTTP
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| PCR Buffer (10X) |
| 1% (v/v) Triton X-100
15 mM MgCl2
500 mM KCl
100 mM Tris-HCl, pH 8.8 at 25°C
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Tris-HCl
dCTP
Oligonucleotide
dTTP
dGTP
dATP
Triton X-100
Potassium Chloride
Mineral Oil
Magnesium Chloride
DNA Polymerase, Taq
Manganese Chloride
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No hints are associated with this bioProtocol
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