1) Thaw all reaction components completely and mix gently to ensure even distribution off all components.
Prepare the reaction on ice in a sterile, nuclease free tube and mix gently after addition of the polymerase.
Collect all liquid at the bottom of the tube by a quick spin.
COMPONENT VOLUME FINAL CONCENTRATION
10X PCR Buffer 5 µl 1X
dNTP Mix (10 mM each) 0.5 µl 0.1 mM each
Primer 1 (10 µM) 1 µl 0.1 µM – 0.5 µM
Primer 2 (10 µM) 1 µl 0.1 µM – 0.5 µM
LabQ Taq DNA Polymerase (5 U/µL) 0.2 µl 1 U
template DNA 1 µl <1 µg
dH2O to 50 µl
2) Keep the reactions on ice until transfer to the thermocycler, then cycle according to these guidelines:
STEP CYCLES TEMPERATURE DURATION
Initial Denaturation 1 94°C 5 minutes
Amplification 30-35 94°C 30 seconds
Tm – 5°C 30 seconds
72°C 1 minute / kb
Final Extension 1 72°C 5 minutes
Hold 1 4°C
3) Analyse the amplification reaction by gel electrophoresis using an acrylamide or agarose gel of appropriate