PCR Amplification of DNA

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.