Protocol P.3

PCR

There are so many variables in PCR analysis that they cannot possibly be addressed here, so I will simply give a representative reaction. Also, see Protocol P.4 for an alternative protocol.

 

Solutions

10X Taq Polymerase Buffer

supplied with enzyme

or:

100 mM Tris pH 9.0

500 mM Kcl

1% Triton X-100

25 mM MgCl2

supplies with enzyme

start with 1.5 mM and titrate up and down to optimize

Primers

dilute to 10 pmols/ml

Template

plasmid or chromosomal DNA or cDNA

25 mM dNTPs

mix equal volumes of each of 4 100mM dNTPs

 

 

Procedure

• Mix the following and place in the PCR machine:

10 ml 10X taq polymerase buffer

0-10 ml 25 mM MgCl2 usually 6 ml is fine

1 ml 25 mM dNTPs

25 ml each oligonucleotide @ 10 ng/ul

0.05 to 0.1 ng plasmid DNA template or

4-10 mg chromosomal DNA

0.5 ml Taq (promega) for a hot start, add after temperature has exceeded the annealing temperature

Q to 100 ml

• Program in the following parameters: 94° 1 minute, 55° 1 minute, 72° 1 minute for 35 cycles and start the program.