From: russell@DOGWOOD.BOTANY.UGA.EDU (Russell Malmberg)
Newsgroups: bionet.genome.arabidopsis
Subject: Non-Radioactive Probes, SUMMARY to daye
Date: 14 Sep 92 12:19:41 GMT
Distribution: bionet


REPLIES to NON-RADIOACTIVE PROBES for ARABIDOPSIS


Attached are paragraphs excerpted from the replies to my query
about using the chemiluminescent detection system for Southerns
and Northerns with Arabidopsis DNA and RNA.

The replies were mostly positive positive but some negative.
There is a lot of interest in this system, as I received many
replies from people who wished to know what the results were.

Thanks to those who replied for their comments!

Russell Malmberg
russell@dogwood.botany.uga.edu


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LISTED in ORDER RECEIVED

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They're great for plasmid blots and colony hybs, but we've found
them too unreliable for lower abundance probing.  And I've never
been comfortable with how you can't tell the "specific activity"
of your probe. There are ways of getting an idea of how much
"label" you've got incorporated (probing a dilution series of
dotted control DNA, e.g.).  And ONCE, we made almost a lifetime
supply of a non-isotopic probe by PCR, but never got that to work
efficiently again.  ...

Tom_Jacobs@qms1.life.uiuc.edu

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We have tried it (as you outlined using "lumiphos" detection) for
a year or so with soybean genomic Southerns.  When it works it's
great, but when it doesn't it is a complete loss (no signal or
awful background). ... With a smaller genomed plant it might work
more routinely, but unacceptable background was usually our
problem.

vance_baird@quickmail.clemson.edu 

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The color reactions are not very good, but I think you probably
can get the chemilum ones to work. ... I use the Boehringer kit,
but we have bought the lumin substrate from Tropix, as it is
cheaper. This last time, I tried labeling by PCR rather than nick
translation and that might be why the results weren't great.

slblowe@uccs.edu

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I have just tried a northern with the GENIUS III kit from BM, the
probes were labeled as riboprobe for in situ hybridization.
Although we did not get success with the in situ, it worked fine
the northern. By the way, we are working with carrot, not
Arabidopsis.

LIN@fcrfv1.ncifcrf.gov

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I am using a Stratagene's Flash Detection System.  It works for 
Southern blot in Arabidopsis.  I did not do Northern blot yet. 
It is not better than 32P, but comparable.  I use random priming
method (followed by spin column) using biotinylated dNTP mix and
T7 DNA polymerase (in fact, it's a USB patented Sequenase so they
do not sell it separately).  One critical point, you have to use
a Flash(TM) membrane they sell.  I tried NitroCellulose filter in
a same batch and it's not working at all. Every procedure is in
the instructional manual.  They guarantee 3 month life, but I
used it for more a year without any problem. It uses avidin-
alkaline phosphotase and lumigen (TM) ppd (chemiluminescence).
When phage screening, a background problem occurred every time.
My impression, it's cheaper than radioactive and easier and
faster.  I recommend it if you want to use it.  But, I do not
have any experience with other brands.  I guess all other brands
would be of equal quality.

tyou@magnus.acs.ohio-state.edu

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During the summer two undergraduates in my group experimented
with the chemilum. system for single copy genes in A. thaliana
genomic Southerns.  The good news is that you get your answer
with 1-8 hour exposures (one hour was usually quite
satisfactory).  The bad news is that the background (speckles
mostly) was high compared to our experience with 32P.  They did
not run 32P controls side by side with the luminescence
experiment because we were trying to avoid undergrads using
radioisotopes.  It is possible that a more experienced person
would have even better results.  They used the GENIUS kit
(what a dumb name).  It is my impression that the cost is fairly
high unless you are able to use the probe over a number of
hybridizations.  I hope that this is useful. ... One day the FDA
will probably tell us that digoxigenin is more dangerous
than 32P.

rob_last@qmrelay.mail.cornell.edu

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I have had reasonable success using the Boehringer Mannheim (BM)
digoxigenin (DIG) system for northerns, Southerns and library
screening with  nucleic acids from plant species with
considerably more complex genomes than Arabidopsis (ie.
jack pine, Eucalyptus). My first piece of advice is to not waste
your time with the chromogenic (ie. NBT/BCIP) detection of DIG
labelled nucleic acids but concentrate on getting the
chemiluminescent (ie. AMPPD, or lumiphos) detection system
working well in your hands.   Also make a judicious choice of
which labelling system you want to use such that it is best
suited to your particular application (eg. random-primed
labelling for long DNA vs 3' tailing for oligos).  Technical
support from BM is great so you should be able to get a pretty
good idea about what it is that you want from them (this is
not to say that BM reps are immune from the sales pitch but I
have found them to be pretty honest with regards to the strengths
and the limitations of the DIG system). ... In general, I've
found the DIG instruction manuals together with a healthy dose of
Sambrook and 'The Red Book' to be pretty well all that is needed
with regards to DIG use.  Nonetheless, I find that the references
indicated within the BM instruction sheets (usually papers
published by BM staffers in journals like Analytical Biochem) to
be very helpful (and sometimes vital,
with the 3' tailing kit for eg.) as well. ...

CAMPBELL@FRCICT81.BITNET

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We've tried the Genius system on Arabidopsis as well as
P.vulgaris genomic Southerns, plasmid Southerns, and phage
screening. I have yet to see anything coherent coming out of this
technique. The chemiluminescence is the worst; extremely high
background. We've had better luck with the NPT detection but
stripping those blots is a pain in the neck. I'm sure the
conditions could be worked out, but at the moment, I have limited
patience.

mchrispeels@jeeves.ucsd.edu

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I would suggest contacting:  Dave Hoisington, Molecular Genetics
Laboratory, CIMMYT, Lisboa 27, Apdo. Postal 6-641, 06600 Mexico,
D.F., MEXICO -- tel. 011-525-954-2100    FAX:  011-525-954-1069 -
- (those numbers assume that you are dialing from the U.S.A.) A
year ago, mail correspondance took 3 to 6 weeks for an Air Mail
letter to reach the P.O Box in Mexico City....I'm not sure if
things have improved.  Dave's EMail address currently bounces so
I didn't give it. The telephone number is directly for CIMMYT in
El Batan and the CIMMYT operators can respond either in Spanish
or English.
For a "preview" of what their non-radioisotopic methods can do
(in maize) you might contact:  Michel Ragot, Dept. of Genetics,
Box 7614, North Carolina State University, Raleigh, NC 
27695-7614 -- tel. 1-919-515-2289   FAX: 1-919-515-3355  
NCORN1@NCSUMVS.BITNET
Michel was in the CIMMYT molecular genetics lab last year doing
that stuff and so, I believe, can be specific on details and
performance. The CIMMYT lab is specifically geared to developing
molbio genetic techniques for maize and wheat improvement that
can be successfully transfered to labs working under the
constraints commonly found once one leaves the USA/Western
Europe/Japan/Australia.

Hope this helps.
         
samodena@csemail.cropsci.ncsu.edu

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We routinely use the Boehringer Mannheim Genius system inour lab.
We have done more than a hundred Southerns and several Northerns
and lambda  and M13 plaque lifts.  We use 1-2 ug digested
Arabidopsis DNA for Southerns for Lumi-phos detection and 3 ug
for color detection.  Both total and polyA+ RNA worked well with
Lumi-phos. Plaque lifts also work better with Lumi-phos than
color detection.
We use several modifications to the kit protocols.
The most important change is the use of maleate buffer instead of
Tris buffer during blocking.  We also increased the stringency of
all washes. ...
Our electronic file transfer is acting up, but I could try to get
it accross. I highly reccomend this system.  It works well, is
easy and has no radioisotope problems.  However, it is not real
cheap.  Please contact me if you  need or want more details.

BTNYLAC@OSUCC.bitnet

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