help! quikchange mutagenesis kit - (Nov/17/2010 )
Help is urgently needed here. THanks a lot in advance.
I am using QuikChange Lightning site-directed mutagenesis kit. I am trying to mutate 4 bases with a 45 bases primer. The primer is designed with the software from Stratgene and is HPLC purified. My plasmid is 8.2 kb and the extension time I used was 5 min. After DpnI digestion, I run the PCR product on the gel, no band around 8kb was found. It is very wierd to see one band around 100 bp and one band around 300 bp. I also did transformation with 5 ul pcr product and 45 ul XL-10 cells. No colony at all. I used correct antibiotic.
Any help from you will be highly appreciated.
Thanks so much for your help.
first, use about 200ng template or more for 'PCR'. actually it is not a PCR reaction. it is linear amplification.
second, try to use 8min or more for extension and 12-18 cycles. you'd better use a high-fidelity polymerase.for your 45nt primer, you can set annealing temperature at 55C or higher.
I often use a 20uL reaction system, 10uL for running gel (if it works very well recently, you may digest directly without running gel),10uL for DpnI digestion. I sometimes have a negative control, that is digest the plasmid with DpnI and transform, and there should be no colony.