ligation problems and electroporation
brad at howard.genetics.utah.edu
Thu Oct 5 12:54:32 EST 1995
In article <450p4g$nnp at edisto.awod.com>, bkodrzyc at awod.com (Bob Kodrzycki)
> We are experiencing ligation problems with "routine" ligations.
>My question is....is electroporation of E. coli superior to the standard
I'm not an expert, but I have used both Heat Shock and Electroporation to
introduce DNA into bacteria. The standard in our lab is to use homemade
DH5alpha that has been made supercompetent by the Hanahan method (I think).
But we clone constructs that can be lethal in a wild type E.coli
so occasionally we need to electroporate. In my hands, electroporation can
be as good as our DH5's (1x10-11) if I am careful about the cells and
a couple rules. 1) Everything must be detergent free for supercompetent
Electro-competents or chemical competent. 2) Catch your O.D.'s at the
right spot. Electroporation may solve your problem.
Your message mentioned that you all are experiencing problems with routine
cloning and transformation. Please don't be offended, but have you
all possible causes of the problem. Things that have troubled me in the
have been: Traces of "bad stuff" in the ligation buffer. It seemed that
ligation buffer was inhibiting transformation. Can you transform
plasmid into your cells? In the same vein, are your cells good, have they
stored in an appropriate freezer? Could anyone be leaving them out to long
ice if there is more that one use per tube? Other people may have other
suggestions, but these are the ones from the top of my head.
Good luck, if you have any questions drop me a line.
P.S. If you decide to electroporate your cells make sure to get rid of the
from the ligation mix. Micro dialysis vs. 50 ml of sterile ddH20 for an
or two is a good method. Also you could precipitate the DNA and resuspend.
Brad Nicholson |"If it worked the first time, it wouldn't be
Department of Pathology | research."...Brad Nicholson
University of Utah |
Salt Lake City, UT 84132 | iligitimi non corborundrum
brad at howard.genetics.utah.edu |
or: (801)-581-4365 | My opinions are solely my own.
More information about the Methods