To help prevent this type of problem, we ususally prepare the DNA used 
for homologous recombination as follows: The plasmid with the 
construct is cut with the appropriate restriction enzymes to release 
the wanted linear piece. Next, we purify the fragement from gel, often 
even twice. We use this fragment as template in a pcr reaction to 
multiply the fragment. Finally the product is purified using a PCR 
purification column and transformed. In our hands, this greatly 
increases the percentage of correct mutants.

Regards,

Leonard Bosgraaf

Biology department
University of Groningen
The Netherlands