In vitro mutagenesis with rec A

Jonathan Bown bownja at
Tue Jul 11 12:55:33 EST 1995

I am trying to clone a section of a gene (in pAA 121) which carries a single 
B.P. substitution into a different vector which carries the same gene of 
interest. Restriction analysis suggests that I should be able to perform a 
simple double digest, ligate and transform. This routine procedure is used 
regularily in our lab, but I am having problems on this occasion e.g. 
purifying the similar sized cut vector away from uncut vector.

I am aware of the possibility of cloning in mutations using the characteristic 
of rec A to insert ssDNA into homol. regions of dsDNA and I have come across 
protocols. I would like to know how widespread the use of this technique is, 
whether it is appropiate for my purpose and what pitfalls to watch for.

Thanks in anticipation,


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