Protocol for Taq 5X Master Mix PCR Reaction

Overview

These recommendations serve as a starting point; in order to maximize amplification the reaction conditions may require optimization (see "Taq 5X Master Mix Guidelines for PCR Optimization" protocol).

Protocol

1. Thaw Taq 5X Master Mix at room temperature and mix well by inverting several times before use.
2. Prepare the following reaction in a thin-walled PCR tube on ice:
   

   Component	25 µl reaction	50 µl reaction	Final Conc.
   Taq master Mix (5X)	5 µl	10 µl	1X
   Upstream Primer (10 μM stock)	0.5 µl	1 µl	0.2 µM
   Downstream Primer (10 μM stock)	0.5 µl	1 µl	0.2 µM
   Template DNA	determined bu user	determined by user	<500 ng
   Nuclease-free water	to 25 µl	to 50 µl

3. Gently mix the reaction and spin down in microcentrifuge.
   If the thermocycler does not have a heated cover, add one drop of mineral oil to the reaction tube to prevent evaporation.
4. Cycling conditions for a routine PCR reaction:
   

   Initial Denaturation	94-95°C	1 minute
   25-40 Cycles	94-95°C 45-70°C 68°C	20 seconds 30 seconds 1 minute per kb
   Final Extension	68°C	5 minutes
   Final Soak	4-10°C