New to map-based cloning, want protocols

Tanya Hooker tshooker at mail.botany.ubc.ca
Wed Sep 17 13:53:58 EST 2003


Hi Martha,

I don't know of any textbook, but for a nice overview of positional cloning,
see Lukowitz et al. (2000) Plant Physiology 123:795-805.

Here are a few pointers from my experience:

-use a decent method for DNA preps - I used a scaled-down Dellaporta method
(to 1.5mL tubes; grinding with plastic pestles for microcentrifuge tubes in
extraction buffer, on ice) (Dellaporta et al. 1983. Plant Molecular Biology
Reports 1:19-21) which worked fine; DNA is stable in the fridge for at least
6 months, so you can use it for all of the markers you want to score.  I
discovered this after trying a simpler, boil in SDS-NaOH, neutralize and use
supernatant method (Klimyuk et al. 1993)- this only gave me about 50%
success rate for the initial PCR reactions, then got worse and worse as time
went on.  I can send you the Dellaporta protocol if you need it.  Some other
people in my lab are using a "Quick and Dirty" method (Edwards et al. 1991.
Nucleic Acids Research 19:1349) that they say works fine for PCR of
fragments under 1.5kb.

-don't freeze and thaw genomic DNA preps - keep them in the fridge (it
degrades if you freeze and thaw it repeatedly)

-Get lots of F2 mutant DNA preps, and save seeds from the plants, so you can
double-check your mutant phenotype later if you need to.  You need at least
500 F2s, but it's a good idea to collect leaves from more than that, in case
you need them.  Just keep the extra tissue in the freezer until its needed.
Don't freeze and thaw this either - you won't get any DNA out of it if it's
been refrozen.

-Make absolutely sure that your mutants are mutants, and not WT-looking.
Otherwise you won't be able to make any sense of your mapping data.

-Map your locus as finely as possible.  Design new markers as needed to nail
down the locus.  Mapping is much easier than trying to complement or
sequence too many genes, if you don't have a really good idea of which gene
might be the right one.  I have learned this from painful experience of
trying to complement my mutant with 19 genes.  It turned out to be the one
closest to the marker for which I had one recombinant, only a couple of kb
away from the marker.

-I have successfully used agarose gels to differentiate between SSLP markers
with as little as 3bp difference on a 100bp fragment.  To get this
resolution, I used a 4% gel with around 1/3 of the agarose being
high-resolution agarose, and a gel with long and narrow slots (as opposed to
squarish slots - you can load more samples per gel this way, but resolution
is worse).  This gave me a nicer picture than the acrylamide gels I have
tried.  I have also used acrylamide gels, though, and they work fine too.

Tanya

***********************************
Dr. Tanya Hooker
Department of Botany
University of British Columbia

tshooker at mail.botany.ubc.ca



----- Original Message -----
From: ""Martha Mullally"" <mrmullal at connect.carleton.ca>
Newsgroups: bionet.genome.arabidopsis
To: <arab-gen at net.bio.net>
Sent: Monday, September 15, 2003 1:50 PM
Subject: New to map-based cloning, want protocols


>  Hi,
>
>  I am new to map-based cloning and am looking for protocols for the
>  technique. Can anyone suggest a good textbook/lab manual which will
>  walk me through the day-by-day methods? I want to use the Col-Ler
>  predicted polymorphisms to map my gene.
>
>  I would really apreciate the help.
>
>  Thanks.
>
>  Martha Mullally
>  mrmullal at connect.carleton.ca
>  Carleton University

---



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