ligation problems and electroporation

Brad Nicholson 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) 
wrote:

> We are experiencing ligation problems with "routine" ligations.  
>snip<
 >My question is....is electroporation of E. coli superior to the standard 
heat shock
 >methods.  
>snip<

Hi Bob,
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 
background, 
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 
follow
a couple rules. 1) Everything must be detergent free for supercompetent 
cells.
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 
eliminated
all possible causes of the problem.  Things that have troubled me in the 
past
have been: Traces of "bad stuff" in the ligation buffer.  It seemed that 
the 
ligation buffer was inhibiting transformation.  Can you transform 
supercoiled 
plasmid into your cells?  In the same vein, are your cells good, have they 
been
stored in an appropriate freezer?  Could anyone be leaving them out to long
on
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.
Brad 

P.S. If you decide to electroporate your cells make sure to get rid of the 
salts
from the ligation mix.  Micro dialysis vs. 50 ml of sterile ddH20 for an 
hour
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.
===========================================================================



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