hyb problems

suter at VAX.MPIZ-KOELN.MPG.dbp.de suter at VAX.MPIZ-KOELN.MPG.dbp.de
Wed Nov 11 06:50:11 EST 1992


+> Help!  I am seeing a very intense background in all my blots, recently.  The
+
+.....stuff deleted...
+>
+> I changed blot (to gene screen plus), changed probe and even used a different
+> pre hyb solution (5x SSC, 5x denhart, 0.5%SDS, 20mM phosphate buffer, 300u/ml
+> salmon sperm DNA, 5mM EDTA), but nothing seems to eliminate the very intense
+> background.  I have even tried washing with water.

+I find GeneScreen Plus requires higher concentrations of salmon sperm DNA for
+blocking than several other membranes.  If the black background doesn't wash
+off then it's covalently bound to your membrane, and the membrane probably
+wasn't sufficiently blocked in the first place.  Try using ca 500ug/ml ssDNA in
+your prehyb mix; I also add a further 1-2mg of ssDNA to my probe when I boil
+it, and then add about 10ml of hyb mix before tipping it into the bag/bottle.
+As for the black membranes, put them away for a few months!
+
+> Jaishree [jaishre at matt.ksu.ksu.edu or jaishre at ksuvm.ksu.edu]
+>
+Good luck!
+
+Martin

a very effecient blocking agent is SDS, we use it at conc of 7 % in our hyb
mix (this is essential for zetaprobe by the way). for washing we use
1 x SSPE, 1% SDS. These rather high concentrations have very relevant effects !
 
cheers,

clemens
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Clemens Suter-Crazzolara, PhD
Max-Planck-Institut fuer Zuechtungsforschung
Abteilung Genetische Grundlagen der Zuechtungsforschung
Carl-von-Linne Weg 10
5000 Koeln 30
Tel. xx49-221-5062.221           fax. xx49-221-5062.21
suter at vax.mpiz-koeln.mpg.dbp.de
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