Dear all Thank you for the responses. This is summary of the replies.
Quick change method: I believe quick change is the standard method. I have utilized a two step method, where two portions of the gene are amplified within the vector (instead of full vector amplification as in quickchange). This process takes about a day longer than quickchange. I have found that if I am unable to make a mutation with quickchange, I can almost always mutate the base(s) using this two step method (without having to order new primers). For example, with a pET vector system I would set up two PCR reactions. The first (called the 'left' reaction) with the T7 primer (forward primer, generally just forward of gene) and with a reverse primer containing my mutation (generally the same reverse primer I would use in quickchange pcr). The second PCR (called the 'right' reaction) would contain the T7Term reverse primer (just down stream of gene) and a forward primer containing my mutation. Then I would gel purify both the 'left' and 'right' amplification products, which should correspond roughly to # bases upstream of mutation and # bases downstream of mutation (+/- for the portion of vector amplified). The next step would be to anneal the two amplification products using PCR. I would put in equal amounts of each product. I would set the PCR to run for about 5 cycles without any primers added to anneal the two products together. Then I would add T7 and T7term reverse primer for a full round of PCR to amplify the full gene with mutation (and up& downstream of gene in vector). Once I have gel purified the gene with mutation, I use appropriate restriction enzymes to digest gene up & down stream (since those sites were amplified in 1st round PCR by using vector primers). Then ligate into vector of choice (or the vector it was already in). Alternative protocol - http://www.biomedcentral.com/1472-6750/8/91 PIPE method: http://www.springerlink.com/content/j02438u4434j257l/#section=92209%26page=1 http://www.ncbi.nlm.nih.gov/pubmed/18004753 http://www.ncbi.nlm.nih.gov/pubmed/18988020 (no DpnI digest require) Phusion method: http://www.finnzymes.com/sequencing_mutagenesis/phusion_site-directed_mutagenesis_kit.html (uses phosporylated primers and no DpnI digest)
