Monday, 19 November 2007

Standard PCR protocol


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 degrees C.
5. Run the following program:
94 degrees C 1 min
55 degrees C 1 min or annealing temperature appropriate for particular primer pair72 degrees C 1 min (if product is <500>500 bp)
for 30 cycles.
Program a final extension at 72 degrees C for 7 min.

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