Dr David E Barton
dbarton at acer.gen.tcd.ie
Fri Jun 16 04:13:33 EST 1995
In article <3rpi8q$gpo at apopi.u-strasbg.fr>,
Francois Nantel <Nantel at titus.u-strasbg.fr> wrote:
>In the following weeks, I am planning to do intensive southern blotting
>of genomic DNA (knockout screen). However, the protocols I have used are
>not perfetct and I have not found two person in the Institute who use the
>same protocol. Therefore, I ask you southern blotters for your favorite
>conditions. Mine are included below:
>DNA size: 5 and 7 kb, 15 micrograms per well
>Gel: 0.7% agarose runned O/N at 40 V
>Gel treatment: 20 min. 0.25 M HCL
>Transfert: O/N alkaline transfert (0.4 M NaOH) on Hybond +
>Probe: 400 bp DNA fragment labeled by random priming
>Hyb buffer: Phosphate-based with 50% formalin
>Pre-Hyb: 1 h
>Hyb: 24 h at 42 oC
>Wash: 2*10 min in 2xSSC+0.1% SDS, RT
> 1*15 min in 0.5xSSC+0.1% SDS, RT
> 1*15 min in 0.1XSSC+0.1% SDS, 65 oC
>The problems I encounter are low specific signal and non-specific (not
>Where should I start?
I'm afraid your life is about to get very complicated! You have probably just
multiplied your problem, because you will get a different answer from
everybody who replies!
Anyway, here goes:
I would increase your hyb temperature (you can go at least as high as 52C)
to get rid of the signal from related sequences, then try washing LESS
stringently to enhance your specific signal.
Try doing it without formamide at 65C - It's nasty stuff and not really
Good Luck, David.
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