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

Nick Jacobsen jacobsen at cf.ac.uk
Tue Aug 27 11:35:10 EST 1996


Hi,
   I have been doing PFGE for a while now and can offer you some tips for 
blotting.After you have run the gel and stained/destained I soak the gel 
in 0.25M HCl for 12minutes (with agitation). Using this method works very 
well for me, over and above UV nicking (with depurination I consistently 
get hybridisation that only needs overnight or sometimes several hours 
for autoradiography). I use BIO-RAD zeta probe-GT membrane which always 
works well and I use the standard SSC transfer method. After blotting I 
fix the DNA to the filter by NaOH soaking and vacuum baking at 80 C. 
These protocols all come with the nylon filters. 

There is a lot of differing opinions with PFGE. It is a very subjective 
technique. The methods above work consistently for me but maybe not for 
you. I would try all suggestions until you find one that suits you,

I hope this helps you
If you need further help, please drop me a line.

cheers

Nick.




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