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

Malay curiouser at ccmb.ap.nic.in
Wed Feb 23 10:11:50 EST 2000


> 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?

Try hybridizing for only 3-4hrs. It solved the same problem in my case.

>
> 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
>
I am looking for some good protocols for cosmid library screening. Can you
share some under-the-table tricks with me?:-)

Malay
> ---

Malay Kumar Basu
Centre for Cellular and Molecular Biology
Hyderabad 500007
I N D I A

Fax: (00-91)40-7171195
Phone: (00-91)40-7172241
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curiouser at ccmb.ap.nic.in



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