Transformation question?

L M lm11 at gpu.srv.ualberta.ca
Thu Jun 5 00:04:14 EST 1997


Dear colleagues,

I'm doing in vitro mutagenesis by Kunkle's method. After I synthesized 2nd
strand and ligated, then tranform host TG1. surprizingly I got no
colonies, but the control plate(normal ds DNA) has a lot of colonies
(>1,000/per ng). 

The synthesis is completed by running a 0.7% agarose gel in the presence
of 0.5ug/mL EtBr). My synthesized DNA band has the same migration as the
supercoiled DNA.
                                       
The ssDNA must contain Uracil, because the titer of my uracil-containning
phagemid in TG1 and CJ236 is very different(>10,000 folds).

Could someone tell me the possible reason?

Thank you in advance

Richard



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