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PCR Amplification of DNA

bullet sterile water
bullet 10X amplification buffer with 15mM MgCl2
bullet 10 mM dNTP
bullet 50 μM oligonucleotide primer 1
bullet 50 μM oligonucleotide primer 2
bullet 5 unit/μl Taq Polymerase
bullet template DNA (1 μg genomic DNA, 0.1-1 ng plasmid DNA) in 10 μl
bullet 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


 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:

bullet94 degrees C 1 min

bullet55 degrees C 1 min or annealing temperature appropriate for particular primer pair

bullet72 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.

This Web page is maintained by Julie B. Wolf, UMBC;
Last updated on 3/2/2010

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