Quickchange is obsolete :) Artem
On Fri, Feb 3, 2012 at 7:56 PM, Roger Rowlett <[email protected]> wrote: > 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_PCR > for details. Allow 1 min/KB each cycle for whole plasmid PCR extension. > > Roger Rowlett > > On Feb 3, 2012 8:29 PM, "Nian Huang" <[email protected]> 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 <[email protected]> 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. >> >> >
