Prepare the following reactions in a PCR tube:
If you are using the 2x Taq Master Mix:
|
Component |
25 μl reaction |
50 μl reaction |
Final Concentration |
|
2X Taq Master Mix(add last) |
12.5 µl |
25 μl |
1X |
|
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) |
|
Template DNA |
~50ng |
~100ng |
<1,000 ng |
|
Nuclease-free water |
~10 µl |
~20 µl |
|
If you are using another enzyme not in a Master MIx:
|
Component |
25 μl reaction |
50 μl reaction |
Final Concentration |
|
10X Polymerase/PCR 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) |
|
Template DNA |
~50ng |
~100ng |
<1,000 ng |
|
Taq DNA Polymerase* |
0.5 µl |
1 µl |
1.25 units/50 µl PCR |
|
Nuclease-free water |
to 25 µl |
to 50 µl |
|
Gently mix the reaction and spin down in microcentrifuge.
Cycling Conditions for a Routine PCR:
|
STEP |
TEMP |
TIME |
|
Initial Denaturation |
95°C |
30 seconds |
|
30 Cycles Melting Temp. Annealing Temp. Extension Temp. |
95°C 56°C 68°C |
30 seconds 30 seconds 1 minute/kb |
|
Final Extension |
68°C |
8 minutes |
|
Hold |
4-10°C |
|
I always use 56 or 56.5C for my Annealing Temperature unless the primer has a large region of random nucleotides, then I might drop it down to 52-54C. This has worked on 99.99% of all of my PCR reactions.
Modified from NEB: https://www.neb.com/protocols/1/01/01/protocol-for-a-routine-taq-pcr-reaction