WANTED- Bombproof Anchor PC

Greg Shen gshen at ucsd.edu
Wed Jun 22 19:51:13 EST 1994


I followed the general method given in the Red Book (section 15.6.1, see 
figure 15.6.1) for amplifying a region downstream of a region of known 
sequence.  I first got a very feint band of interest when I ran out (on a 1%
regular agarose gel in TBE buffer) a small sample of the PCR reaction 
products (using a forward primer that was specific for a region of known 
sequence, and a poly-T reverse primer that had 3 restriction sites tacked on 
to it).  

I then took the rest of the initial PCR reaction products, EtOH precipitated 
the solution (skipping the phenol:chloroform extraction), resuspended it 
in TE buffer, and ran it out on a 1% metaphor (lo melt) gel (in TAE buffer), 
cut out the band of interest, and diluted it 1:10 with TE buffer.  I used 1 
ul of this "1:10 band" directly as the template for the second PCR round (
using a total reaction volume of 50 ul, and performing 30 cycles).

I got a very intense band. 

The key was using Taq plus a tiny bit of pfu (contained in Stratagene's Taq 
extender or diluted yourself just before use - this is referred to in 
Barnes paper: PNAS March 94, where he gets 35kb PCR products using this 
combination).  Try 1 part Taq to 1/32 part pfu.  I hope that helps!



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