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site directed mutagenesis

mutagenesis

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3 replies to this topic

#1 student47

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Posted 17 April 2014 - 05:35 AM

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??



#2 bob1

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Posted 17 April 2014 - 01:32 PM

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.

#3 phage434

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Posted 18 April 2014 - 04:10 AM

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



#4 student47

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Posted 15 May 2014 - 07:39 AM

cheers guys, it worked..







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