PCR
1. Reaction recipe
Mix the following components (make sure the stocks you use are at the same concentrations as those in table):
1 |
38 ul of deionized sterile water |
Used to bring up to 50 uL |
2 |
5 ul of Thermo Pol Buffer 10 X New England Biolabs |
|
3 |
1 ul of dNTP mix (25 mM of each) |
|
4 |
1 ul of Upstream primer |
0.1 mM stock, 100 pmol final |
5 |
1 ul of Downstream primer |
0.1 mM stock, 100 pmol final |
6 |
1 ul of DNA template (15 ng/uL) |
15 ng final recommended |
7 |
1 ul of Taq DNA polymerase (New England Biolabs, 5units/ul) |
2. Program setting:
Step |
Temp. C |
Time (min) |
Description |
1 |
95 |
5 |
Initial denaturation |
2 |
95 |
1 |
Denature |
3 |
Tm* |
1 |
Anneal |
4 |
72 |
1 |
Elongate (generally, 1kb/min) |
5 |
Goto step 2 |
29 cycles |
Cycle (25-35 only, otherwise enzyme decay causes artifacts) |
6 |
72 |
10 |
Final elongation |
7 |
4 |
hold |
|
8 |
End |
*the calculation of Tm
Tm=4*(G + C) + 2*(A + T) - 5
(usually between 50-65 C)