Ideas behind PCR optimizations: In order to optimize PCRs, we need to ask the following questions:
Generic PCR Recipe
For a 10ul reaction:
|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 (10uM)||0.75ul|
|Reverse Primer (10uM)||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
- 98° for 30 seconds (Use 5 minutes in the case of colony PCR to lyse the cell)
- 98° for 10 seconds (Denaturing temperature)
- Annealing Temperature for 30 seconds (see section below)
- 72° (Extension temperature; Use 30 seconds per 1kb of expected product)
- Go to step 2, and repeat 34 times (so, 35 cycles total)
- 72° for 5 minutes
- 12° hold
Use this link to get the melting temperature of a primer, using Phusion polymerase and 500nM primer concentration.
The annealing temperature for PCR is then this melting temperature that is systemically modified.
- Primers greater than 20 nucleotides in length anneal for 10–30 seconds at 3°C above the melting temperature of the lower melting temperature primer. If the primer length is less than 20 nucleotides, an annealing temperature equivalent to the melting temperature of the lower primer should be used.
If you are working with new primers, use this protocol to find the ideal annealing temperature.
- Initial test: Try a single 10ul reactions and an annealing temperature of 3 degrees below melting temperature of primers.
- If you do not see a single, correctly-sized band, switch to a gradient PCR using 8 tubes 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)