Hybe conditions

Klaus.Matthaei at anu.edu.au Klaus.Matthaei at anu.edu.au
Thu Oct 15 18:40:11 EST 1992


G'Day Netters

I have had a number of enquiries about the hybe conditions that I use
(which were worked out in the Reed Lab some years back) so I thought that I
would post them to avoid confusion. 

Note that these are for positvely charged membranes such as Hybond N+ after
alkaline transfer. 
 DO NOT USE THESE HIGH TEMPERATURES FOR NITROCELLULOSE.

DNA Probe DNA Target
2xPE, 7% SDS, 1% BSA hybe at 70 degrees or 
50% formamide, 7% SDS, 1% BSA at 43 degrees (To get the SDS into the hybe
dissolve the SDS in the formamide).
Wash down to 0.5x SSC, 1% SDS at 70 (or 0.2xSSC at 65).

DNA Probe RNA Target
50% formamide,2xPE, 7% SDS, 1% BSA at 50 degrees 
Was down to 0.2xSSC, 1%SDS at 70

Oligo probes
5xPE, 7% SDS, 1% BSA at the TH calculated from the oligo from
4x(G+C)+2x(A+T)-5 degrees C.
Wash down to 5xSSC, 1% SDS at the TH.

RNA Probe DNA Target.
50% formamide, 2xPE, 7% SDS, 1% BSA at 50 degrees.
Wash down to 0.2xSSc, 1% SDS at 70 degrees (if the background is high rinse
the membrane with 2xSSc and incubate in 1 ug/ml RNase A in 2xSSC for 15 min
and rewash in preheated 0.2xSSC 1% SDS etc).

RNA Probe RNA Target.
60% formamide, 2xPE, 7% SDS, 1% BSA at 60 degrees
Was as for RNA Probe DNA Target.

Washes are usually for 15 minutes only and a typical protocol is:
Rinse membrane after hybe briefly at RT in 2xSSc 0.1% SDS.
Wash 15 min RT 2xSSC 0.1% SDS with vigorous agitation.
Was in preheated hot wash as above and vigorous agitation.
Rinse in 2xSSC 0.1% SDS, Blot with 3mm, wrap in Saran wrap or similar film
(Caution some plastic films give you Saran-Zap from static electricity and
other background problems,  The Australian Glad-Wrap is also OK) and
expose.

Good Luck, Klaus
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Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei at anu.edu.au

"If all else fails : Read the instructions"
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