FAQ: How should I set up an amplification reaction using OneTaq® DNA Polymerase?

We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (94°C).

COMPONENT
25 μl
REACTION
50 μl
REACTION
FINAL
CONCENTRATION
5X OneTaq Reaction Buffer*
5 μl
10 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)
OneTaq 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
 
*OneTaq GC Reaction Buffer and High GC Enhancer can be used for difficult amplicons

**For amplicons between 3–6 kb, use 2.5–5 units/50 µl rxn

Notes: Gently mix the reaction.  Collect all liquid to the bottom of the tube by a quick spin if necessary.  Overlay the sample with mineral oil if using a PCR machine without a heated lid.

Transfer PCR tubes from ice to a PCR machine with the block preheated to 94°C and begin thermocycling:
STEP
TEMP
TIME
Initial Denaturation 94°C
30 seconds
30 Cycles 94°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