hybridization mixes

suter at VAX.MPIZ-KOELN.MPG.dbp.de suter at VAX.MPIZ-KOELN.MPG.dbp.de
Wed Feb 19 07:07:20 EST 1992


dear netters,

sorry if you get this message twice:

Axel Themmen wrote (among other things):
< Could you give me the complete recipe of the hybmix? This method could save
< me a lot of label and hybmix. Also, what is the volume you user for 20 phage
< filters (I assume lifts from a 15 cm agarplate?).

my high stringency hyb mix is:

50 % formamide; 0.12 M Na2 HPO4 (pH7.2); 0.25 M NaCl; 7 % SDS (w/v);
1 mM EDTA; 0.01 mg/ml denatured Salmon Sperm DNA (sambrook).
In fact, this is the same mix (except for the salmon sperm) used for Biorads
Zetaprobe membranes, but it works perfectly well for genescreen or
nitrocellulose. The SDS dramatically lowers the background (this I
have tested for genescreen). Sorry, I don not have another reference for this,
nor did I use this method with prepunched holes, as somebody asked me.

The low stringency buffer recipe I do not have here, but I could look this up
For twenty phage filters of 15 cm, I would use two tubes, each with
5 to 10 ml hyb mix (I do not know this of the top of my head): the important
thing is that all the filters can get evenly wet during rotation. I generally
have probes of say 1 x 10E6 per ml.
Most people are afraid that signals from one filter are transferred to the next:
this I have however never noticed (I fix the DNA to NC by baking at 80 degrees
(without vacuum) for 30 min)

ciao, clemens

 ###############################################################
 # dr. c. suter-crazzolara    mpi for plantbreeding      koeln #
 # tel --49-221-5062.221      fax --49.221.5062.213            #
 # e-mail: suter at vax.mpiz-koeln.mpg.dbp.de                     #
 ###############################################################
  #             konstantijn had een hobbelpaard               #
   #              zonder kop en zonder staart,               #
    #              zo reed hij de wereld rond               #
     #              zo maar in zijn blote...               #
      #####################################################



More information about the Methods mailing list