Help: 2 Plasmid Electroporation /Infection

TJ tjc at itsa.ucsf.edu
Wed Apr 10 14:36:45 EST 1996


I was hoping for suggestions as I am having difficulty getting two 
plasmids into E.coli at library efficiences.  I am hoping to add a M13 
derived vector to cells containing a pUC/pBR3222 plasmid, or vice versa.

Electroporating the M13 derived vector has been at low efficiency so 
far, making double electroporations very inefficient.

The cells containing these plasmids were less electrocompetent, less 
than 10^6 col/ ug of the second plasmid.  Therefore, this approach would 
require many electroporations to get a large library.

I next looked to make the M13 derived vector infectious by growing with 
helper phage, but had too much cross over with the helper phage.  I am 
now trying this in a Rec minus strain.

I was hoping to electroporate in one plasmid and then add the other 
plasmid through lambda infection but was unable to locate a non lysing 
system.

Please let me know if you have had similar experiences or any 
suggestions.  Thanks
tj

t j cradick   
ucsf dept of immunology 
please reply to:tjc at itsa.ucsf.edu                           





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