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PCR Amplification M.2: PCR AMPLIFICATION OF DNA

Materials:

sterile water
10X amplification buffer with 15mM MgCl2
10 mM dNTP
50 uM oligonucleotide primer 1
50 uM oligonucleotide primer 2
5 unit/ul Taq Polymerase
template DNA (1 ug genomic DNA, 0.1-1 ng plasmid DNA) in 10 ul
mineral oil

1. Combine the following for each reaction (on ice) in a 0.5 ml tube:

10X PCR buffer	10 ul
Primer 1	1 ul
Primer 2	1 ul
dNTP	2 ul
template DNA and water	85.5 ul
Taq Polymerase	0.5 ul

2. Prepare a control reaction with no template DNA and an additional 10 ul of sterile water.
3. Add 70-100 ul mineral oil (or 2 drops of silicone oil) to each reaction.
4. Place tubes in a thermal cycler preheated to 94oC.
5. Run the following program:

94oC 1 min
55oC 1 min or annealing temperature appropriate for particular primer pair
72oC 1 min (if product is <500 bp), 3 min (if product is>500 bp)

Program a final extension at 72oC for 7 min.

6. Electrophorese 10 ul on an agarose gel.