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??