Wittrup: PCR

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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.

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