DNA Extractions....

PAB6DC at VAX.BIO.LEEDS.AC.UK PAB6DC at VAX.BIO.LEEDS.AC.UK
Mon Aug 2 07:23:14 EST 1993


Some time ago, I asked for help in getting pure DNA from dried leaves of various 
_Ficus_ species - all I could get was what seemed to be a rubber ball! (lots of 
latex), and badly degraded DNA. Several people wrote back with advice - thank 
you all. We tried everything suggested, except the use of CsCl gradients (problems 
with getting small enough rotors, and cost), but none of them worked for our 
tissue. We now have a method that seems to work, which uses a CTAB-based 
buffer, followed by the Promega Magic-Miniprep resin to clean up the DNA. We 
get restrictable DNA, still somewhat degraded, although I think that is because of 
the state of the starting tissue. Below are extracts from the various suggestions - 
they may be of use for other plant species that people are having trouble with.

Thank you all again for your suggestions - it was fun trying them ....!


David Coates
Dept. Pure and Applied Biology
THE University of Leeds
Leeds LS2 9JT
UK

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Dear David:
I have used the DNA preparation protocol described in Behringer, F.J., and
Medford, J.I. A plasmid rescue technique for the recovery of plant DNA
disrupted by T-DNA insertion.  Plant Mol. Biol. Rep. 10:190-198 with great
success.  Although I have only used the method for preparation of DNA from
Arabidopsis, I wonder whether it might be useful for your situation.  There is
no precipitation of DNA until after the DNA has been purified on a cesium
gradient.  If you decide to use this protocol, I would suggest spinning the
preparation at 12,000 xg after you add the cesium, but before you spin in the
ultra, just to get rid of the worst of the junk that will otherwise stick to
the side of the ultra tube (I use a vertical rotor).
Best of luck, and welcome to the amazing (frustrating?) world of plant
secondary metabolism.
Clint Chapple
Michigan State University

*******
Sender: carries at gov.nrel

Dear David,
I was alble to remove polysaccarides from Arabidopsis RNA preps by 
centrifuging the ethanol precipitaion *before* adding sodium acetate. The
polysaccarides precipitate, leaving RNA in solution. Then, adding salt
brings down the RNA. Perhaps this would work with latex as well.
Carrie Schneider

*****
From: pickett at edu.uoregon.molbio
Subject: Re: DNA extraction
To: PAB6DC at UK.AC.LEEDS.BIO.VAX
Message-id: <01GYEN7EI9WY8Y5C28 at OREGON.UOREGON.EDU>
Content-transfer-encoding: 7BIT


Hi,  I don't know if you've tried an SDS based extraction method, but I
have used a protocol derived from the Dellaporte protocol on Arabidopsis
and Tobacco with fairly good success.  It utilizes a potassium acetate
precipitation of SDS to remove carbohydrates and proteins, I don't know if
it will also do a good job of bringing down latex.  I have also used a very
quick and dirty CsCl based DNA purification based on June Medfords
published in the Plant Molecular Biology Reporter May 1992 issue.  We have
had to slightly modify both protocols for tobacco,  I will be happy to
e-mail you our protocols if you think they'll help.

Bryan Pickett  

*******
From: Terence Brown <Terence.Brown at uk.ac.mcc.mailhost>
Original-Sender: Terence.Brown at uk.ac.mcc.mailhost

I'm not sure if this will help but your problem sounds similar to one we have
had with archaeological material that we are trying to extract DNA from. Often
the DNA preps contain nasty brown contaminants of uncertain origin. The DNA
rarely works in PCR or cloning in this state.

CTAB has not helped us much. I suggest you try cleaning up the initial 
extracts with polyvinylpolypyrrolidone - not the PVP you use in hybridization
analysis but PVPP. Our method involves mixing 1 g of plant (or whatever)
material + 0.3 g PVPP in 5 mls phosphate buffer pH 8.0 + 0.2 mM ascorbic
acid (latter as an antioxidant). The PVPP is subsequently precipitated at the
interface by phenol-chloroform. You can use your standard procedure once you
have done the PVPP pre-treatment.

PVPP is supposed to bind phenolic compounds so whether it will work for you
is a bit questionable. But it is pretty cheap so maybe worth a try.

Best wishes,

Terry Brown,
Department of Biochemistry and Applied Molecular Biology,
UMIST, Manchester M60 1QD.

******
From: J R Beeching <J.R.Beeching at uk.ac.bath.gdr>
To: pab6dc at uk.ac.leeds.bio.vax
Subject: Latex and DNA

I extract DNA from cassava (Manihot esculenta) which also contains latex.
I've found that the Dellaporta method works fine.  The ref is:
Dellaporta SL, et al. (1983) A plant DNA minipreparation: Version II.
Plant Molecular Biology Reporter 1:19-21.

If you can't get hold of the ref, and want to give it a try I can e-mail
you a version.

All the best,

John Beeching.

*******
From: "Sarah.Gilmour" <22676SJG at edu.msu>
Subject: DNA extraction
In-Reply-To: The letter of Thursday, 20 May 1993 1:04pm ET

David,
I can't answer your question, but maybe Katrina Cornish can as she works on
the rubber producing plant (I can't spell  it but it's pronounced why-ulee).
She works at the USDA in Albany, California.
Sarah Gilmour

*********
From: Elizabeth.A.Pease at EDU.Dartmouth (Elizabeth A. Pease)
Subject: Re: DNA extraction
To: PAB6DC at uk.ac.leeds.bio.vax
Sender: Elizabeth.A.Pease at EDU.Dartmouth

I am not sure if I had the same problem or not, but when I isolated
Arabidopsis DNA from frozen tissue my sample was very brown and undigestable. 
I solved this by spooling the DNA out of an ethanol precipitate instead of
spinning and drying (using a capillary tube that was held over a hot flame to
seal the end and make a sort of hook,I  just spin it in the ethanol, salt,
DNA mix and the DNA sticks to it forming a sort of "snotball") .  Then with
the DNA "snot ball" hanging off the glass rod I dipped it in 2 or 3 rinses of
70% ethanol (two or three tubes containing the ethanol and just progressively
dip it in each) finally resting it in an empty tube (rod and all) to dry for
a few minutes then resuspend in a large volume of TE rinsing off the rod. 
This was still slightly yellow however digested very well.

I hope this helps.



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