COMPONENT |
25 μl REACTION |
50 μl REACTION |
FINAL CONCENTRATION |
10X Reaction Buffer |
2.5 μl |
5 μl |
1X |
10 mM dNTPs |
0.5 μl |
1 μl |
200 μM |
10 μM Forward Primer |
0.5 μl |
1 μl |
0.2 μM (0.05-1 μM) |
10 μM Reverse Primer |
0.5 μl |
1 μl |
0.2 μM (0.05-1 μM) |
Taq DNA Polymerase |
0.125 μl |
0.25 μl |
1.25 units/50 μl PCR |
Template DNA | variable |
variable |
<1,000 ng |
Nuclease-Free Water | to 25 μl | to 50 μl |
Transfer PCR tubes from ice to a PCR machine with the block preheated to 95°C and begin thermocycling:
STEP |
TEMP |
TIME |
Initial Denaturation | 95°C |
30 seconds |
30 Cycles | 95°C 45-68°C 68°C |
15-30 seconds 15-60 seconds 1 minute/kb |
Final Extension | 68°C |
5 minutes |
Hold | 4-10°C |