Blotting DNA from Polyacrylamide

RPSCOTT RSCOTT at IRRI.CGNET.COM
Sun Feb 14 03:25:33 EST 1993


Doan Nguyen (nguyend at carr106.alleg.edu) writes:
>I NEED TO KNOW IF ANYONE HAS ATTEMPTED TO BLOT DNA FROM NATIVE
>POLYACRYLAMIDE GELS.  CAN IT BE DONE BY CAPILLARY ACTION OR 
>SHOULD IT BE DONE BY ELECTROBLOTTING?

There is a procedure for electroblotting DNA from polyacrylamide
gels to nylon by D. M. Coen in Current Protocols in Molecular 
Biology. Capillary transfer apparently doesn't work well with
PAGE-separated DNA as mentioned in that article. When you blot
try using postively-charged nylon instead of regular ones. In
our experience, charged membrane retains nucleic acids more
efficiently and requires no UV-treatment or baking for sample
fixation. You may be hesitant however to use positive nylon 
for fear of background problems during hybridization. But you
need not as long as you block the membrane efficiently. We have
tried Hybond-N+ (Amersham) for Southern blot hybridization
with digoxigenin-labelled probes and found no background with
either luminescent (AMPPD) or color (NBT/BCIP) detection methods 
for the hybrid signals. Likewise, we find our signals crisper
and more intense on charged than on regular nylon.

Electroblotting is quite rapid but if you insist on capillary
transfer you might as well switch to sieving agarose gels or
agarose composites with either NuSieve (FMC) or Synergel 
(Diversified Biotech). I assume  of course that you are dealing 
with relatively small fragments of DNA. We have tried both
Synergel and NuSieve composites for resolving PCR products. We
have found Synergel binaries as superior than those of NuSieve 
in terms of resolution efficiency of closely spaced fragments and the
improved translucency which affords clearer documentation after
ethidium staining. Synergel composites however are rather flimsy
to handle but may be used for capillary Southerns.

Best wishes on your work and good day. 

Scott :-)

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RPScott
Genetics Laboratory
Plant Pathology Division
International Rice Research Institute
PO Box 933, 1099 Manila, Philippines
e-mail:  rscott at irri.cgnet.com

"Advice is an uncertain gift." -- W. Jeffrey (Reader's Digest)
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