Wittrup: PCR
Materials
Taq polymerase (usually 5 U per 0.001 mL) and 10X buffer
MgCl2
forward primer
reverse primer
DNA template
PCR grade water
dNTP mix or each of dATP, dCTP, dGTP, dTTP
2.5 M betaine (optional)
DMSO (optional)
Mix the components of each 0.050 mL reaction on ice:
The final concentration of each of the reagents are as follows:
10X buffer---------------1X
MgCl2--------------------final concentration 1.5 to 2.5 mM
forward primer----------0.5 uM
reverse primer----------0.5 uM
dNTP---------------------10 mM each of dATP, dGTP, dTTP, dCTP
DNA template------------typically 10 ng
water----------------------add water to 0.049 mL volume
Taq Polymerase---------2.5 to 5 Unit (usually 0.001 mL)
The use of DMSO and betaine usually improve specificity of the annealing reaction. If the use of DMSO and betaine are desired, the final concentration of DMSO and betaine should be 3% (v/v) and 1 M.
Proceed with thermal cycling conditions outlined below:
Denature-------1 min at 94 degree Celsius
Anneal----------1 min at the proper annealing temperature (depend on the primers)
Extend----------1 min for each 1 kb at 72 degree Celsius
Repeat for 30 cycles (typical amplification)
Final extension for 5 to 10 min at 72 degree Celsius.