Help! my colony blots are lighting up like a christmas tree

R. Jayakumar jakku at mrna.tn.nic.in
Wed Feb 16 12:29:12 EST 2000


I am sorry I forgot to mention.  The probe is actually a PCR amplicon
developed using a higly specific primers for that particular gene which I
need to pull out.  The amplicon was sequenced and confirmed to be the
expected fragment.  The PCR was done against the same bacterial total DNA
from which I made the library.  The probe was screened against both
bacterial DNA and the E.coli total DNA.  While the former gave the expected
positive signals, the later was negative as expected, in a dot blot.
Moreover the PCR amplicon was gel eluted using a commercial elution kit  and
then labelled and used as a probe.  After labelling, the probe was purified
on a Sephadex column.  As you can see ther is no chance of plasmid or any
other foreign DNA contamination.
    I believe it could be due to bacterial debris still sticking on to the
colony lifts on the colony lifts.
    What do you say?

jayakumar

----- Original Message -----
From: Frank O. Fackelmayer <Frank.Fackelmayer at uni-konstanz.de>
To: <methods at hgmp.mrc.ac.uk>
Sent: Wednesday, February 16, 2000 10:06 PM
Subject: Re: Help! my colony blots are lighting up like a christmas tree


> "R. Jayakumar" wrote:
>
> > Hi
> >    Somebody should really help me out with this problem.  I did colony
lifts
> > of about 1000 clones onto Both nitrocellulose and Nylon (N+) colony lift
> > membrane discs and did a normal hybridisation with my DNA probe labelled
> > with p32 using the GIBCO BRL's random primer labelling kit.  But nearly
all
> > my colonies lighted up, with both the membranes I used.
> >     Can anybody tell me where the mistake is?  For your information, I
> > followed the colony lift technique given in Maniatis manual.  As
> > recommended, I have also given prewashes of the blot before doing
> > hybridization.
> >     Any suggestion will be most welcome
> > thanking you in advance
> >
> > cheers
> > jayakumar
>
> Hi Jayakumar,
> Sounds like your probe hybridizes to the bacterial genome or a region in a
> plasmid. The simplest guess would be that the probe you labelled was
> contaminated with plasmid DNA that now lights up all bacteria that have
the
> plasmid...
>
> Hope this helps,
> Frank
>
>

---




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