We prefer to do MEGAWHOP PCR and use 1-5 uL of the DPN digested PCR product. This is extremely reliable with commercial competent cells. See http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Recombinant+DNA+Protocols#Site_directed_mutagenesis_by_Megaprimer_Quick_Change_MEGAWHOP_PCRfor details. Allow 1 min/KB each cycle for whole plasmid PCR extension.
Roger Rowlett On Feb 3, 2012 8:29 PM, "Nian Huang" <huangn...@gmail.com> wrote: > Just a reminder. Quickchange is not PCR. It is linear amplification. It is > very hard to see a band in the gel if you follow the standard protocol. > > Nian > > On Fri, Feb 3, 2012 at 12:14 PM, Fred <ccp4bb.l...@gmail.com> wrote: > >> Hi CCP4 list, >> Thanks everyone who have answered my post concerning to mutagenesis. >> From quick reading most of the answers, the following seems to be a >> consensus: >> 1) Do not concentrate your PCR product; >> 2) Too much DNA and/or impurities like salts or whatever can inhibits >> transformation; >> 3) Purify your PCR product before transformation if possible or use 3 of >> 4 microL of it. This is more or less the amount of DNA showed in the >> uploaded image. >> Kind regards, >> Fred >> >> P.S.: I'll let you know the results. >> > >
