WANTED- Bombproof Anchor PC
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
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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