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
