PCR Amplification of DNA
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sterile water | ||||||||||||
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10X amplification buffer with 15mM MgCl2 | ||||||||||||
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10 mM dNTP | ||||||||||||
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50 μM oligonucleotide primer 1 | ||||||||||||
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50 μM oligonucleotide primer 2 | ||||||||||||
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5 unit/μl Taq Polymerase | ||||||||||||
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template DNA (1 μg genomic DNA, 0.1-1 ng plasmid DNA) in 10 μl | ||||||||||||
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mineral oil (for thermocyclers without a heated lid
1. Combine the following for each reaction (on ice) in a 0.2 or 0.5 ml tube:
2. Prepare a control reaction with no template DNA and an additional 10 μl of sterile water. 3. If the thermocycler does not have a heated lid, add 70-100 μl mineral oil (or 2 drops of silicone oil) to each reaction. 4. Place tubes in a thermal cycler preheated to 94EC. 5. Run the following program: |
94EC 1 min
55EC 1 min or annealing temperature appropriate for particular primer pair
72EC 1 min (if product is <500 bp), 3 min (if product is >500 bp)
for 30 cycles.
Program a final extension at 72EC for 7 min.
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