Generic PCR

Generic PCR Recipe

For a 10ul reaction:

Reagent	Volume
Phusion Reaction Buffer (5X)	2.0ul
There are two types. Initially, try the High GC buffer (green cap) for Streptococcus, and the High Fidelity (red cap) for other purposes.
dNTPs (Nucleotides; 40 mM total)	0.2ul
Forward Primer (2uM — 1:50 dilution of the concentrated, 'blue cap' tubes)	0.75ul
Reverse Primer (2uM — 1:50 dilution of the concentrated, 'blue cap' tubes)	0.75ul
DNA template (< 50ng/ul)	1.0ul
Phusion Polymerase (5U/ul)	0.1ul
Molecular-grade Water (from refrigerator)	5.2ul

PCR Machine Protocol, Phusion polymerase

1. 98° for 30 seconds (Use 5 minutes in the case of colony PCR to lyse the cells)
2. 98° for 10 seconds (Denaturing temperature)
3. Annealing Temperature for 30 seconds (see section below)
4. 72° (Extension time; Use 30 seconds per 1kb of expected product)
5. Go to step 2, and repeat 34 times (so, 35 cycles total)
6. 72° for 5 minutes
7. 12° hold

Annealing Temperature

Use this link to get the suggested annealing temperature of a primer pair, using Phusion polymerase and 500nM primer concentration.

If you are working with new primers, use this protocol to find the ideal annealing temperature.

• Initial test: Try a single 50ul reactions with the suggested annealing temperature
• If you do not see a single, correctly-sized band, switch to a gradient PCR using 8 tubes each with 10ul across 10 degrees.
  • If there are more than one band in the initial test, use the original annealing temperature as the lowest temperature in the gradient
  • If there are no band in the initial test, use the original annealing temperature as the highest temperature in the gradient

Generic PCR Recipe with DMSO

Problematic PCR's? Try using 3%-10% DMSO. This will then change the annealing temperature, necessitating a gradient PCR across 15 degrees.

10% DMSO (1ul in 10ul reaction)