In vitro mutagenesis with rec A

Jonathan Bown bownja at bham.ac.uk
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,

Jon



More information about the Methods mailing list