Mutagenesis to make 45bp deletion - (Mar/03/2006 )
I would like to generate a 45bp deletion in a plasmid using mutagenesis - I have tried several pairs of primers containing the mutation with no success. I can generate 1 bp insertions and deletions so the kit is fine. Is such a large deletion possible to generate? Any advice for designing primers?
U do not have to use mutation to do deletion. what you can do:
design a pair of primers to make sure that sperate at the space you want to delete, but at reverse direction (oppsite) on the same plasmid, you want to bring the same restriction enzyme sites on each primer. Then amplify the whole plasmid using good polymerase by PCR. digest the PCR product and do ligation, you got it!