Materials:
sterile water
10X amplification buffer with 15mM MgCl2
-10 mM dNTP
50 μM oligonucleotide primer 1
50 μM oligonucleotide primer 2
5 unit/μl Taq Polymerase
template DNA (1 μg genomic DNA, 0.1-1 ng plasmid DNA) in 10 μl
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
10X PCR buffer 10 μl
Primer 1 1 μl
Primer 2 1 μl
dNTP 2 μl
template DNA and water 85.5 μl
Taq Polymerase 0.5 μl
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 94°C.
5. Run the following program:
94°C 1 min
55°C 1 min or annealing temperature appropriate for particular primer pair
72°C 1 min (if product is <500 bp), 3 min (if product is >500 bp)
for 30 cycles.
Program a final extension at 72°C for 7 min.
