Best Mutagenesis Strategy --- SUMMARY

the End jgraham at bronze.ucs.indiana.edu
Fri Nov 19 15:44:57 EST 1993


Hong,

Your strategy does seem straight forward, however I would be concerned about 
PCR of an entire plasmid (2-3 Kb). The notion of a smaller circular DNA 
may work, but it sounds like a potential problem, but perhaps will not be 
in the quantities necessary for a PCR target. Isolation of the correct form 
may be the most difficult step.
In case you go with a Kunkel:

In reviewing my own experience with the Kunkel procedure, I find the most 
difficult problem was creating templates free of endogenous small fragments
capable of "self-priming" the template. In addition, a hard spin for 
ten minutes, then removal of the top half of the phagemid containing 
supernatant and another hard spin was necessary to prevent whole cells 
from carrying ds wt  plasmids into and through the procedure.

Coincidently, another group right there at Baylor (Lebovitz and Osei-Frimpong)
recently reported improving efficiencies from 3% to 100% in the Kunkel
mutagenesis on the same template by cleaning up the ss template preparation 
with an Exo III treatment (Biotechniques vol.14 (4) 1993). This apparenlty 
removes those troublesome small self-priming fragments for a ss template 
prep. My attempt  to clean up these template with an alkaline agarose gel 
purification was a dismal failure :)


Good luck,

Jim
J. Graham



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