Site directed mutagenesis - Of 20 kb plasmid (Mar/10/2003 )
Has anyone carried site directed mutagenesis of a 20 kb plasmid? I have tried it using Quick change kit according to their protocol but it is not working. I don't get any colonies after transformation. The extension time I am using is 1 min/kb. I have tried 2 min/kb also with the addition of extra enzyme after 7 cycles. I were using 1 mM dNTPs. With 2 min extension time I get a smear and with 1 min I don't get anything. With less dNTPs (500 uM) and at annealing temperature of 68 deg instead of 60 I get multiple bands which are smaller than 20 kb . Does any one have any suggestions or conditions for carrying out that experiment?