Hi Fred: For the mutated plasmids, it generates the nicked dna, so the transform efficiency will be lower compared to the parental plasmids. And that is the reason why people usually use super competent cell to transform these nicked plasmid. It seems ok to me to get 2 or 14 colonies from the nicked plasmid. You just need ONE. And usually for these mutagenesis PCR, the success rate is pretty high. I suggest you to sequence these colonies. And you can get it.
Yu Xiaodi > Date: Tue, 7 Feb 2012 10:17:43 -0200 > From: ccp4bb.l...@gmail.com > Subject: Re: [ccp4bb] [RESUME] [OFF-TOPIC] Site-Directed Mutagenesis > [OFF-TOPIC] > To: CCP4BB@JISCMAIL.AC.UK > > Hi CCP4 list, > Thank you very much for additional messages and references. > Here goes the image of the "PCR" product before digested and after > digested and cleaned. > http://ompldr.org/vY29jbA > The results of the transformation of 3 microL (90 ng) of > non-mutated/paretal plasmid gave hundreds of colonies; 3 microL of Dpn1 > digested sample gave two colonies only; and transformation of 3 > microL(90 ng) of cleaned product gave 14 colonies. > So, if the amplification is not abundant, chances are that home made > competent cell will not be transformed with the digested product. Don't > want start another discussion but, is there any reason for differences > in the transformation efficiency between the parental and the mutated > (cleaned) plasmids? > All the Best, > Fred > > Em 03-02-2012 16:14, Fred escreveu: > > 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.
