| Contributor: |
The Laboratory of Donald Rio at the University of California, Berkeley
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| Methods that use a high fidelity polymerase combined with high input template DNA concentration and low cycle number yielding virtually error free PCR (it is still recommended that you sequence your products). |
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Prepare 50 μl reactions
200 to 400 ng Template (plasmid) DNA
5 μl of 10X Vent Buffer
4 μl of 2.5mM dNTPs (200 μM final concentrations)
50 pmole oligonucleotide primers (1 μM final concentration)
0.5 μl Vent Polymerase (1 Unit final concentration)
Added ddH2O to 50 ul
Top reaction with approximately 2 drops of mineral oil
15 Cycles As Follows
94°C 4 min
94°C 1 min (melting)
55°C 1 min (annealing)*
72°C 1 min (extension)**
* Use an annealing temp of 2 to 4°C less than the Tm of the oligonucleotide with the lower Tm (see Hint #1).
Tm = 2°C(A-T base pairs) + 4°C(G-C base pairs)
OR
Tm = 69.3 + 0.41(%G/C) - 650/number of nucleotides
** Extension time is based on the size of the amplified fragment approximately 1 min per 1,000 base pairs.
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| dNTPs |
| 2.5 mM dGTP
2.5 mM dCTP
2.5 mM dTTP
2.5 mM dATP
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| Primers |
| 50 pmoles oligonucleotide
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| Vent Polymerase |
| 2 Units/μl Vent Polymerase
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| Template DNA |
| Concentration 200 to 400 ng
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| Plasmid DNA |
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| Vent Buffer |
| 10X Vent Buffer (from N.E.B.)
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Vent Polymerase
Mineral Oil
dCTP
dTTP
Oligonucleotide
dGTP
dATP
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1. Remember the Tm is calculated for nucleotides in the oligonucleotide COMPLEMENTARY to the template DNA. Don't include restriction sites of G/C clamps.
2. Larger oligonucleotides (40mers to 70mers) with the standard 17 to 21 nucleotides of complementarily sometimes amplify poorly. Invariably when Taq polymerase is used instead of Vent Polymerase the contributor of this protocol detect efficient amplification. Lowering the Tm and varying the Mg2+ concentration may improve efficiency with Vent Polymerase.
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