Southern Blotting of Pulse Field Gels.... HELP!!?

Pascal Cousin Pascal.cousin at chuv.hospvd.ch
Mon Aug 26 05:15:10 EST 1996


Graham Dellaire wrote:
> 
> Hello all,
> 
> I am going to be blotting DNA resolved by pulse field electrophoresis
> and I am worried about what protocol to use...
> 
> Should I use a regular southern protocol as for genomic DNA?  Or are
> there some modifications  I should employ?  What is a good membrane for
> this kind of transfer?
> 
> I would like to hear about your experience with this technique...
> 
> Any and all suggestions needed and appreciated,
> 
> G. Dellaire
> Div. of Exp. Medicine
> McGill University
> 
> dellaire at odyssee.net
Hi,

I have done alot of pulse-field southerns. I compared UV nicking and
acid-base size reduction:UV nicking of large DNA is more reproducable
than acid-base depurination. 
I also compared types of transfers:
Alkali transfer onto neutral nylon membranes works better than SSC
blotting.
Below is the detailed protocol I use, which works for fragments up to
800 kB.
One last remark: Do not over-load gels, 1-5 10^6 nuclei  per lane is
sufficient.

 
Pulse field gel treatement.

Staining destaining.
· Stain 15’ in 500 ml EtBr 1 ug/ml in H2O.
· Destain 1 hr in 500 ml H2O.
· Photography gel.
Denaturation.
· UV irradiation in Stratalinker at 800 100 mJoule/cm2
· Denature gel in 500 ml NaOH 0.4 M / NaCl 1 M for 45’
Transfer.
· Transfer gel onto HyBond membrane (Amersham) with 0.4 M NaOH / 1 M
NaCl, O/N with 500g weigh
· Neutralize membrane in 0.5 M Tris pH 7.2 / 1M NaCl 2 x 10’ RT (250 ml)
· Crosslink DNA to membrane 2 hr in oven at 80°C.

Good luck

		P. Cousin



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