14 Polymerase Chain Reaction
211
Procedure
1. In advance prepare the following on ice:
ln advance
a. dNTPs (Perkin Eimer Cetus): prepare a stock solution (lmM each
dNTP):
dATP {10 mM)
75 J..ll
dCTP {10 mM)
75 J..ll
dGTP {10 mM)
75 J..ll
dTTP {10 mM)
75 J..ll
ultrapure water
450 J..ll
total volume
750 J..ll
b. primers: dilute to 20 J.!M ( = 20 pmol/J.!l)
c. DNA template: dilute to 10 5 -10 6 template molecules/SJ.!l (3 x 10 5 template molecules is equivalent to 1J.!g ofhuman genomic DNA, 10 ng of
yeast DNA, 1 ng of E. coli DNA, and 0.3% of an Ml3 phage plaque)
2. This protocol provides for a 100 J.!l reaction, but can easily be scaled down Standard PCR
to a total volume of 10 J.!l. For all PCR reactions performed with the same protocol
primer pair, a master mix is generated containing every component except the DNA template. For n reactions a master mix for n+ 1 reaction
volumes should be prepared to have sufficient material available.
In a 1.7 ml Eppendorf prepare a master mix on ice by pipetting n+ 1 times
the volume for one reaction combining the following components:
Component
Volume for
Final
Stock
Amount
one reaction
concentration concentration
a. PCR buffer
10 J..ll
1x
10x
b. dNTPs
5 J..ll
50 J..LM each
1 mM each
5 nmol each
dNTP
dNTP
dNTP
c. Primer pair
2 X 1J..Ll
0.2 J..LM each
20 J..LM each
20 pmol each
d. Taq. polymerase 0.5 J..ll
0.025 U/J..ll
5 U/J..Ll
2.5 u
e. Ultrapure H20
77.5 J..lla
a The later addition of 5 J..Ll of DNA template will make a total volume of 100 J..Ll per
reaction (see below).