Follow-up : hybridization enhancement

Karl Fischer tyr-2 at bones.biochem.ualberta.ca
Thu Jan 29 10:38:09 EST 1998


Hi everyone,

A couple of weeks back I was asking about hybridization enhancement.
Decided to post this so interested parties could benefit from the
information.

The responses I was getting centered around addition of CTAB (1 mM) to the
hyb solution (ref PNAS 1991, vol. 88, pp. 8237 - 8241 by Brian W. Pontius
and Paul Berg).

Decided to give it a try. 

Dot-blot hybridization - samples were sera (10 ul/spot) which were alkali
denatured on the membrane (HyBond N), neutralized then UV crosslinked. 

Conditions - 6X SSC, 6.5% SDS, 1 mM CTAB - block 1 hour at 65C then add
probe (1 X 10^7 cpm, sp. act. 2.5 X 10^8 cpm/ug) - hyb three hours at 65C.


Washes - 5 minutes, RT, 2X SSC-0.1% SDS; 2X 15 minutes, 65C, 2X SSC-0.1%
SDS; 5 minutes, RT, 0.2X SSC-0.1%SDS; 2X 15 minutes, 65C, 2X SSC-0.1% SDS.
Expose to phosphoimaging plate for 6 hours. Result - sensitivity less than
1 pg/dot. Made for a slightly longish day but was happy with the results.
Blot was as clean as a 7% SDS-6X SSC hyb solution that I usually use.

Would I use it again to "push the (time) envelope"? You bet!!

My thanks to all who responded.

Cheers

Karl the hepB guy



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