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 94EC.
5. Run the following program: | 94EC 1 min
55EC 1 min or annealing temperature appropriate for particular primer pair 72EC 1 min (if product is <500 bp), 3 min (if product is >500 bp)
for 30 cycles.
Program a final extension at 72EC for 7 min.
上一篇：Detection of Alu by PCR 下一篇：Polymerase Chain Reaction
|
Detection of A