Stuart Brown browns at ccu.umanitoba.ca
Wed Jun 24 15:15:47 EST 1992

In article <35452.timmer at ccwf.cc.utexas.edu> timmer at CCWF.CC.UTEXAS.EDU (@ccwf.cc.utexas.edu, Richard T. Timmer) writes:
>Fellow Netters:
>We have recently cloned a number of proteins from
>Arabidopsis and wheat, and we are now in the process of "attempting" to
>carryout the Southern analysis for each of our clones.  However, we are
>having very little success...the problems are as follows:
>   1.  Background:  We have tried both random primed 32P-labelled probes and
>Millipores chemiluminescent SolarPlex method; our blots were obtained from
>gels in which we had run out approx. 10 micrograms of genomic DNA and blotted
>by alkaline transfer to nitrocellulose or nylon (respectively for the two
>methods of detection).
>   2.  Problem:  Even after 2-3 wks of exposure for the 32P-labelled probes
>we are hard-pressed to see any bands--  so, no detection.
>Any suggestions on what variables to consider to improve our detection
>(e.g. amount of genomic DNA, probe/detection considerations, transfer
>methods, etc.)
>Thanks in advance for your help.
>Richard Timmer
>Dept. of Chem. and Biochem.      (phone)    512-471-4562
>The Univ. of Texas at Austin     (fax)      512-471-8696
>Austin, TX  78712                (e-mail)   timmer at ccwf.cc.utexas.edu

I have found that the single most important factor in genomic Southerns
is the type of membrane used, followed by the hybridization conditions
(this is assuming that you have high quality genomic DNA and a pure 
probe labelled to reasonablly high specific activity).  I have had the
best success with positively charged nylon membrane (GeneScrene, HyBond,
ZetaProbe) and hyb. solutions containing 2-4% SDS, salt, and sheared
non-specific DNA.  

No way should you be using 2-3 week exposures with 32P.  After about 4
days your radioactive decay means that you are adding less than 1/2 of
the intensity of the 1st day for each additional day - If you can't see
anything useful after 2 days, weeks of expsure aren't going to help.

Try changing your membrane and using a different hyb solution.
Also, assay your DNA used to make probe for purity, and count
specific activity after labelling.

	Luck  -Su

Stuart M. Brown                               Postdocs rush in where wise men
U. of Manitoba, Dept. Plant Science, Canada       Fear to tread. 
browns at ccu.umanitoba.ca        
				Disclaimer?? Sure: "That Disc is Mine!"

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