Missing plasmid after miniprep

Jon Nakamoto jnakamot at ucla.edu
Thu Sep 26 03:48:17 EST 1996


Could your host bacteria be full of revertants (the amber mutations on the
p3 episome can spontaneously revert, especially when under pressure of
high tetracycline/ampicillin concentrations, where revertants are favored
over non-revertants which are depending on the supF in pcDNAI for their
survival)? I think that Invitrogen specifically recommends using low
amp/tet concentrations (lower than you might use for most other
selections) to avoid this problem.

Jon

Jon Nakamoto, MD
Asst Clin Prof of Pediatrics/Endocrinology
UCLA
jnakamot at ucla.edu


In article <527fet$2mt at woodstock.socs.uts.EDU.AU>, Nick dos Remedios
<nick at bio.uts.edu.au> wrote:

> 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

-- 
Jon Nakamoto
jnakamot at ucla.edu



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