bckraev at aeolus.ethz.ch
Fri Oct 7 21:04:20 EST 1994
I use (since 1985) a hybridization buffer that is 0.5 M NaPO4 (molarity
for Na+!)-1% BSA-7%SDS. It is great for all kinds of membranes, but I did
notice a problem of spotty background with some brands of SDS ( e.g., Fluka).
I always use Bio-Rad SDS for the hybridization buffer, and any other SDS
for washes, which are made in 0.1M NaPO4-1% SDS ( from the same 1M NaPO4
stock). Most nylon membranes have no background after 2x10 min washes at RT.
Higher temp. is necessary only to achieve a certain stringency. As for the
carrier DNA, I would still add it because it suppresses spurious signals of
occasional probe homology to high copy sequences ( so, the carrier lowers
background on the tracks, not on the membrane). In addition, probes are
stable in the buffer at room temperature (radioactive - until they decompose
due to radiolysis, non-radioactive have been monitored for 6 months so far).
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