PCR - glasgowlab/home GitHub Wiki

Q5 polymerase

Standard super-high-fidelity polymerase for most PCRs. We buy a master mix.

25 µl rxn

• 12.5 µl Q5 2x MM (final conc. 1x)
• 1.25 µL 10 µM primerF (final conc. 0.5 µM)
• 1.25 µl 10 µM primerR (final conc. 0.5 µM)
• 0.5 µl template (<1000 ng)
• 9.5 µl water

Phusion polymerase

This is a great polymerase: fast and high-fidelity. We can express and purify it ourselves. Use with GC buffer and 0.5 µl DMSO if necessary.

25 µl rxn

• 0.5 μl 10 mM dNTPs (200 μM each)
• 0.5 μl 10 μM primerF (400 nM each, Finnzymes says 500 nM but 200-1000 nM okay)
• 0.5 μl 10 μM primerR
• 5 μl 10x HF buffer
• 0.5 μl template (1pg-10ng plasmid, 50-250ng genomic)
• 17 μl water
• 0.5 μl polymerase