Missing plasmid after miniprep

Nick dos Remedios nick at bio.uts.edu.au
Mon Sep 23 21:02:05 EST 1996


Here's my problem ('hope someone can help), 

I am COS cell cloning a receptor gene using a monoclonal antibody to pan
a cDNA library in pcDNAI (similar to pcDM8). I have done three rounds of
panning and have transformed the plasmid DNAs back into the appropriate
strain of bacteria (MC1061/p3). I get a couple of hundred colonies on my
LB-amp-tet plates. I minipreped 24 colonies (larger ones) using the
Maniatas alkaline lysis method (1.5 ml culture vol) and digested with
appropriate RE's (10% DNA was used for digest and run on agarose gel).
Result was that of the 24 samples, only one contained vector (which also
had a 1.8 kb insert). The other 23 lanes showed no plasid DNA.

One sample worked perfectly which indicates the problem is not nuclease
contamination. I am perplexed that there appears to be no plasmid in
23/24 samples even though they grew in presence of amp+tet (both on
plates and in O/N cult.). Minipreps did give DNA pellets but this
could've been RNA and contaminating chromosomal DNA. 

Due to the unusual antibiotic selection system in pcDNAI, this sort of
thing may be common but I am not sure. Any help will be appreciated (if
replying could you please email me a copy).

Nick dos Remedios
nick at bio.uts.edu.au



More information about the Methods mailing list