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.
