FAQ: How should I set up an amplification reaction using OneTaq® Quick-Load® 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 25μl
REACTION
50μl
REACTION
FINAL
CONCENTRATION
5X OneTaq Quick-Load Buffer* 5 μl 10 μl 1X
10 mM dNTPs 0.5μl 1μl 200μM
10 μM Forward Primer 0.5μl 1μl 0.2μM (0.05-1μM)
10 μM Reverse Primer 0.5μl 1μl 0.2μM (0.05-1μM)
OneTaqQuick-Load DNA Polymerase** 0.125 μl 0.25μl 1.25 units/50 μl PCR
Template DNA variable variable <1,000 ng
Nuclease-Free Water to 25 μl to 50μl  

*OneTaq Standard Reaction Buffer can also be used
**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