Southern Blotting of Pulse Field Gels.... HELP!!?
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
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
Below is the detailed protocol I use, which works for fragments up to
One last remark: Do not over-load gels, 1-5 10^6 nuclei per lane is
Pulse field gel treatement.
· Stain 15 in 500 ml EtBr 1 ug/ml in H2O.
· Destain 1 hr in 500 ml H2O.
· Photography gel.
· UV irradiation in Stratalinker at 800 100 mJoule/cm2
· Denature gel in 500 ml NaOH 0.4 M / NaCl 1 M for 45
· 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.
More information about the Methods