PCR Amplification of DNA

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 degrees C. 5. Run the following program:

94 degrees C 1 min

55 degrees C 1 min or annealing temperature appropriate for particular primer pair

72 degrees C 1 min (if product is <500 bp), 3 min (if product is >500 bp)

for 30 cycles.

Program a final extension at 72 degrees C for 7 min.