Help! Problem with Northern Blots!->Membrane swaps & Church hyb

Bradley Turner bsturner at mbcrr.harvard.edu
Thu Apr 10 13:27:35 EST 1997


Hello Joy,

There has recently been discussion in this group and in the literature
about manufacturers changing the formulations of their nylon membranes
causing high background on blots.  Below I've included some of the emails
that contain the references to these discussions.  I recently spoke with
one of the suppliers about this and they did say that a change in their
nylon membrane formulation led to unacceptably high background hybridization
and that using "Church's hybridization buffer" as discussed below would
eliminate this.

I think that you can access the TiBS articles via the WEB at

http://www-lmmb.ncifcrf.gov/~pnh/

(all kudos to Paul Hengen)

I hope this helps,
Brad Turner


**************************************************************
		    Bradley Turner
	        Beth Israel Deaconess
                    Medical Center

Harvard Medical School		617-667-1215 phone
Division of Gastroenterology	617-667-2767 fax
Room Dana 536			bsturner at mbcrr.harvard.edu
330 Brookline Avenue		bturner at bidmc.harvard.edu
Boston, MA 02215, USA		turner at sprcore.bidmc.harvard.edu
**************************************************************



JOY'S ORIGINAL QUESTION--------------------------------------------------

>From jbuckle at cs.mun.ca Wed Apr  9 14:24:35 1997
To: methods at net.bio.net
From: Joy Buckle <jbuckle at cs.mun.ca>
Subject: Help! Problem with Northern Blots!
Date: Wed, 9 Apr 1997 15:12:31 -0230
NNTP-Posting-Host: ganymede.cs.mun.ca
Mime-Version: 1.0


I have been carrying out Northern blots for the last six months and the
lab tech here has been doing them for 2 or 3 years.  Things up until now
were fine but in the last month or so we have been producing blots that
are literally half black (full of radioactivity) and half normal. For
example, today I developed a membrane from a large gel.  The bottom lanes
were perfect but the top half was simply black.  It was impossible to even
determine where the lanes were. This is why it is so puzzling.  We are
using nylon membranes and prehybridizing for 4 hours in Denhardt's, SDS,
SSC and Salmon Sperm DNA.  This is being carried out in a hybridization
oven @ 42 C in the bottles.

Only one membrane is contained in each bottle. We then hybridize overnight
with the DS probe @ 42 C again.  The one thing I did notice this time was
that when I rolled the membrane and the nylon mesh into a tight spiral to
place it into the bottle, the part of the membrane that turned out to be
black was the part that was directly exposed to the solutions (i.e. facing
the interior of the bottle).  

This was working fine before using all the same recipes for the solutions
used.  Anyway have any ideas what could be wrong?  We've asked evryone
here in the dept but no one has seen or heard anything like this before.
We even contacted Mandel, the producers of NYTRAN- the nylom membrane that
we are using)  but to no avail.


Any suggestions?   Please email jbuckle at ganymede.cs.mun.ca

Thanking you in advance,
Joy




END OF QUESTION---------------------------------------------------------

SOME EMAILS ADDRESSING THIS TOPIC---------------------------------------

>From pnh at ncifcrf.gov Thu Apr  3 11:42:12 1997
To: methods at net.bio.net
From: pnh at ncifcrf.gov (Paul N Hengen)
Subject: Re: Best Hybridization Solutions for Southerns
Date: 3 Apr 1997 16:15:50 GMT
NNTP-Posting-Host: cockleberry.ncifcrf.gov
X-Newsreader: TIN [UNIX 1.3 950824BETA PL0]

paul gold (paulgold at MO.NET) wrote:

> Our lab is gearing up to do Southerns in order to analyze genomic DNA
> from potential homologous recombinant ES cells for a knockout.  There
> are a lot of mixes, both commercial and in manuals, that claim maximum
> sensitivity with low background.  We would greatly appreciate feedback
> from those of you that have a favorite hybridization solution.  Thanks.

If you plan on using the positively charged nylon membranes such as
Nytran Plus, Hybond N+, Magnacharge, BioDyne A, etc., you are probably
better off using Church's formula for the hybridization. The people on
this group say that it reduces the background considerably, especially
with the new Hybond N membranes that have changed formulation recently.
If you use a different hybridization solution, avoid adding formamide.
See the section on "Churchgoers" in this article:

@article{Hengen1996Octtibs,
author = "P. N. Hengen",
title = "Methods and reagents - Purification of {GST}
fusion proteins",
journal = "Trends in Biochemical Sciences",
volume = "21",
number = "10",
pages = "400-401",
month = "oct",
year = "1996"}

@article{Church1984,
author = "G. M. Church
     and W. Gilbert",
title = "Genomic sequencing",
journal = "Proc. Natl. Acad. Sci. USA",
volume = "81",
pages = "1991-1995",
comment = "church hybridization solution",
year = "1984"}

--
*******************************************************************************
* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
* - - -  Methods FAQ list -> ftp://ftp.ncifcrf.gov/pub/methods/FAQlist - - -  *
* -  TIBS column archive -> http://www-lmmb.ncifcrf.gov/~pnh/readme.html - -  *
* - The BEST Molecular Biology HomePage -> http://www-lmmb.ncifcrf.gov/~pnh/  *
*******************************************************************************


>From TA at Piercenet.com Fri Apr  4 11:20:24 1997
X-Authentication-Warning: news.acrux.net: [207.51.206.137] didn't use HELO protocol
Date: Fri, 04 Apr 1997 10:21:11 -0800
From: Technical Assistance <TA at Piercenet.com>
MIME-Version: 1.0
To: Bradley Turner <bsturner at mbcrr.harvard.edu>
Subject: Re: Best Hybridization Solutions for Southerns AND NORTHERNS?
Content-Transfer-Encoding: 7bit

The church buffer is commercially available from Pierce as Prod # 28394.
-- 
Technical Assistance - Pierce Chemical Co
mailto:TA at piercenet.com
http://www.piercenet.com


END OF EMAILS-----------------------------------------------------------

ANOTHER REFERENCE------------------------------------------------------

In the TiBS article above, there is a reference to another TiBS article
that specifically talks about the issue of membrane SWAPS. That reference is:

TiBS 20:522-523, 1995

END OF REFERENCE-------------------------------------------------------




END OF INCLUDED MESSAGES---------------------------------------------------



More information about the Methods mailing list