site directed mutagenesis - (Apr/17/2014 )

hi,

 i have a 200 bp insert in a 7.3 kb plasmid and am using agilent technologies mutagenesis kit to produce a mutation in 5 sequences in the middle of the insert sequence. the kit works by running a pcr using primers that has the mutated sequence in it which get incorporated during the pcr. as per the instruction of the kit, primers were desigened complimentary to each other , conaining mutation required and pcr was run. i never get colonies, and when i do it does not have the mutation but the original sequence instead. the kit is working fine, i checked. the original dna is digested after the pcr using dpn1. there is nothing else i can think of apart from trying a variation of the concentration of the pcr reagents. does the sequence of addition of pcr products matter? since the kit is working fine, only other varibles are the dna template ( i tried a few different batches) and the primers ( i dont think it can be a problem, its ordered from outside and sequences are correct ) . any other ideas??

You can do a slight modification of the kit, which is to use primers that don't completely overlap - they should overlap for about 1/2 their length in the region where you want the change to be. This should be more efficient than doing conventional SDM al la Qickchange.

Bob1 means that the overlap should be on the 5' end of each sequence. A 3' overlap will fail immediately.

cheers guys, it worked..