PCR Using Q5® Hot Start High - Fidelity DNA Polymerase ( M0493 ) .
Set up the following reaction :
Component25 µl Reaction50 µl ReactionFinal Concentration5X Q5 Reaction Buffer5 µl10 µl1X10 mM dNTPs0.5 µl1 µl200 µM10 µM Forward Primer1.25 µl2.5 µl0.5 µM10 µM Reverse Primer1.25 µl2.5 µl0.5 µMTemplate DNAvariablevariable < 1,000 ngQ5 Hot Start High - Fidelity DNA Polymerase0.25 µl0.5 µl0.02 U / µl5X Q5 High GC Enhancer ( optional ) ( 5 µl ) ( 10 µl ) ( 1X ) Nuclease - Free Waterto 25 µlto 50 µl .
Gently mix the reaction .
Collect all liquid to the bottom of the tube by a quick spin if necessary and overlay the sample with mineral oil if using a PCR machine without a heated lid .
Transfer PCR tubes to a PCR machine and begin thermocycling .
