RFLP frequency(replys)

[N.Yoshida]

Thank you for everyone who replyed to my original message.
I would like to try more enzymes.
Thank you.


Here are the replys.
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We have had quite good luck with RFLPs, scoring perhaps an RFLP with 10% of the
enzymes.  Using a 1-2 kb piece we usually get 1-2 polymorphisms from 10
enzymes.

CAPS also works quite well.  Because the probe being tested is usually smaller,
we test more enzymes.  We routinely test 30-40 enzymes and almost always get a
polymorphism.  Twice, however, we  have had to resort ot RFLPs, and there we
got polymorphisms fairly easily.

Rob McClung
Dartmouth College

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Dear Nobu, we had a case where a Ler marker line (the one with visible
markers on both arms of chromosome 3) had in fact a stretch of Col-0 DNA.
So consider a cross to another ecotype, or one to a different Ler line, as
an additional option which has to be taken seriously.



        Andreas Bachmair
        Inst. of Botany
        Univ. of Vienna
        Rennweg 14
        A-1030 Vienna Austria
        Tel.: (+43) 1 79794 174
        Fax:  (+43) 1 79794 131
        e-mail: bachmair at S1.botanik.univie.ac.at

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The chances of finding a RFLP have varied enourmously in our experience.
However, we recently had trouble with 2 different clones, both of which
gave us La/Col polymorphisms with CfoI, but not 16 other enzymes, so CfoI
might be worth a try!

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* Brenda Winkel Shirley                                    *
* Assistant Professor           540-231-3013 (phone)       *
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You may get a good result if you have tried 50 Enzymes.
 
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>1.How often can you find RFLPs when you test a few enzymes(6 bases cutter)
> for a probe(1-10kb).

Test 6 to 10, in several cases you will be lucky, but it greatly varies.
Sometimes 40 or more are necessary.


> I would like to ask the same question regarding CAPS(0.5-2kb) with 4-5
> bases cutter enzymes

Same answer. On the other hand we just failed with a 3 kb PCR product to
find an RFLP testing > 50 enzymes.
What we do now is to amplify both Col/ LER and sequnce the POCR products
with the PCR primers from both end. A commercial company here reads approx.
500 bp of high quality. So you may find polymorphisms. (in the mentioned
case even that did not help so far.
>
>2.When I can not obtain any RFLPs with a few enzeymes, I try more enzymes
>  or longer prebe. Any other strategies can be taken?

Is answered above.

Good luck,


Tony Schaeffner

***********************************************************

PLEASE NOTE THE NEW ADDRESS (STREET NAME AND ZIP CODE)

Dr. Tony Schaeffner
Institut f. Biochemie
Ludwig-Maximilians-Universitaet Muenchen
Feodor-Lynen-Str. 25
D-81377 Muenchen
Germany

Fax    +49-89-7401-7314
phone  +49-89-7401-7311
email  schaeff at lmb.uni-muenchen.de

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YOSHIDA,Nobumasa
Bioscience Laboratory, Mitsui Petrochem.Ind.LTD
TEL:+81-8275-3-9211    FAX:+81-8275-3-8811
E-mail:yoshida at mipec.co.jp
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