Direct PCR of plaques

B VISSER * x2818 Plantkunde * pbv at rs.uovs.ac.za
Thu Apr 20 01:21:29 EST 1995


Hi

This is just to say thanks for all the help offered with my problems.  I Now 
get excellent results using the following:

+- 1 000 000 individual clones
73 pmoles of each primer
1.66 mM MgCl2
161 micromolar dNTP's
1.5 U Taq pol from Advance Biotechnologies

Before putting everything together, the pfu's are first heated to 70oC for 
10 minutes, cooled and the rest of the stuff are added.  The cycles are 
then as follows:

94oC for 2 min (1X)

94oC for 1 min
65oC for 1 min
72oC for 2 min (35X)

Above concentrations were obtained using an excellent paper for optimization 
by Cobb and Clarkson, 1994 Nucleic Acids Research 22: 3801-3805.

Thanks again.

Botma Visser




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