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

R. Jayakumar jakku at mrna.tn.nic.in
Wed Feb 16 15:30:29 EST 2000


HI
  I had already done exactly as you said with both the NC as well as with
the nylon blots according Maniatis, and as you said in the same trough with
the blots rubbing against each other, on a rotating shaker at 42 C in a
special washing buffer containing edta and SDS for 2 hours.  It did not seem
to be of any help.  I followed zeta probe protocol for hybridizations for
nylon blots and normal protocol for NC.  Stringency washes was done at 65C
    Now where did I go wrong?
sincerely
jayakumar

----- Original Message -----
From: Hiranya S. Roychowdhury <hroychow at nmsu.edu>
To: "R. Jayakumar" <jakku at mrna.tn.nic.in>
Cc: <methods at hgmp.mrc.ac.uk>
Sent: Wednesday, February 16, 2000 11:48 PM
Subject: Re: Help! my colony blots are lighting up like a christmas tree


> At 05:29 PM 2/16/00 -0000, R. Jayakumar wrote:
>  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
>
> You are exactly right! Colony blots tend to light up indiscriminatey due
to
> the bacterial cell debris remaining on the filter. There will always be
some
> background due to this but the signal-noise ratio will increase several
fold
> by doing what I used to do.
> I used to get good results by washing a bunch of those filters together in
a
> small container (in which the filters would be able to rub against each
> other constantly) using the minimum volume of buffer. What actually
happens
> is that the remainder of the bacterial cell debris sticking to the
membrane
> rubs off during this. You need to change the wash buffer frequently. Try
> it... you may be surprised.
> This has been my experience in cosmid genomic library screening. I used to
> grow the colonies directly on the membranes... but that is another
protocol...
>
>
> Dr. Hiranya Sankar Roychowdhury
> GENE LAB/ EPPWS
> New Mexico State University
> Las Cruces, NM 88003
> Ph. (505) 646-5785
> hroychow at nmsu.edu
>
>

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