I did site-directed mutagensis with pcr (two different pCR product and combine together and amlify template with 5 ans 3 primer containing restriction sites, PCR product size is correct), but I try to clone site-directed pcr product cut by xhoI and spe I ligated into vector pCMV6 cut by same restrion enzymes. I got clone for 3 different mutations but I never get clone for 3 more different mutations.
I also did site-directed on doubl stranded plasmid and ligation, but never get mutated plasmids.
Is there any suggestions for site directed mutagenesis ( any recommendation) and any comment ligation?
Thank you for advance