PCR of Alpha-Lytic Protease DNA

PCR Solution (all reagants given in final concentrations):

1. 200 µM dNTPs
2. 10 ng template
3. 500 nM of each primer
4. 1 U of Polymerase
5. Polymerase buffer
6. 1.5 mM Mg⁺⁺ (if polymerase buffer doesn’t already contain Mg⁺⁺)
7. 10% DMSO

PCR Program:

1) 30 seconds @ 94° C (melting temp.)

2) a) 30s @ 94° C

b) 60s @ 60° C (annealing temp.)

c) 60s @ 72° C (extension temp.)

3) Repeat step two 29x

4) 5 min @ 72° C

Notes:

1. DMSO is the key ingredient in the PCR cocktail. This reagant allows proper base-pairing of the primers on the high GC content alpha-lytic DNA. Without it (in my experience) you get smaller, nonspecific products.
2. You can use many different polymerases for the PCR. I have successfully used Taq, Pfu, and PfuTurbo. Taq is fast whereas Pfu is slower but has higher fidelity and PfuTurbo (from Stratagene) has both advantages (I have had the best success with this enzyme).
3. You may want to change the extension temperature in your program according to the optimum temperature of the enzyme that you are using.
4. Annealing temperature can play a large role in whether your PCR is successful, so it’s often helpful to use the gradient feature on the Robocycler on your first PCR attempt. A recommended started annealing gradient is 52° C-66° C.