How should I set up an amplification reaction using Crimson Taq DNA Polymerase?

We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (95°C).

COMPONENT	25 μl REACTION	50 μl REACTION	FINAL CONCENTRATION
5X 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)
Crimson Taq DNA Polymerase	0.125 μl	0.25 μl	1.25 units/50 μl PCR
Template DNA	variable	variable	<1,000 ng
(25 mM MgCl₂ Solution)*	(1.5 μl)	(3 μl)	1.5 mM
Nuclease-Free Water	to 25 μl	to 50 μl

*MgCl2 addition is only required when using Mg-Free Reaction Buffers.

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 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

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