Contaminating plasmid

Paul N Hengen pnh at fcs260c2.ncifcrf.gov
Thu Jan 13 18:36:36 EST 1994


ray at leicester.ac.uk wrote:

>> A number of us have had problems with a plasmid which seems to
>> contaminate our plasmid stocks. It's ampicillin resistant and
>> has a linear size of around 2.2 kb. The strangest aspect is the
>> lack of sites for common restriction enzymes. It has an SstI (SacI)
>> site but lacks sites for BamHI, HindIII, KpnI, PstI, EcoRI, SalI
>> and EcoRV.
>> 
>> It often appears as a faint band in preps of plasmid DNA from
>> constructs in pUC and Bluescript type vectors.
>> 
>> Can anybody suggest what it might be? I'd be very grateful for
>> suggestions.

Christian Geltinger <geltinger at gsf.de> wrote:

: Hi netter, We had the same problem with Bluescript-preparations in E.coli XL1
: in our lab a year ago, and fortunatly, it disappeared as it came. We speculated
: that it might be some sort of ssDNA which would explain the noncutability of
: that plasmid.  This might be due to a leaky f1 origin of bluescript.

Do you mean nicks created at f1 ori that would make a single-stranded version
of the pUC or Bluescript plasmid? I'm confused about what a "leaky" f1 ori is.

R. A. KELLN <KELLN at Meena.CC.URegina.CA> wrote:

| How sure are you that it is a contaminating plasmid per se?  Given the
| frequency of this happening to others who are using pUC, I can't help but
| wonder if the commonality here is that you are all using an alkaline rapid
| lysis procedure and what you are seeing is, in fact, denatured supercoiled DNA
| which is refractory to digestion by most restriction enzymes...[rest deleted]

What's the difference between single-stranded plasmid DNA and denatured
supercoiled DNA? One is linear and the other is not?

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
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