Subject: Re: Genomic DNA: Polysaccharide contamination

From: [email-masked] (Anne Gillen)
Date: 11 May 1996 01:08:28 GMT

You have my sympathy.  I worked with apricot and had the same 
problem.  I have three suggestions.
1.  Let the leaves sit in the fridge for a day or two.  They may use up 
    their polysaccaride reserves.
2.  Unfortunatley I do not have the protcoll that I developed (based 
    on Saghai-Maroof et al)with me but it was an SDS extraction buffer, 
    cholorform:isoamyl alcohol (24:1) extaction.  I used a salting out step
    (ammonium acetate)before the extraction step which I picked up from
    Dellaporta et al 1983 (I think this is the right reference).
3.  Cesium chloride band the DNA.  Expensive, time consuming but it works. 
    We do it for all our peach DNA. 

Sorry for the vague references but I know if I do not reply now I never 
will.

Good luck,

Anne

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From: [email-masked] (Chris Kafer)
Date: Sat, 18 May 1996 22:29:40 GMT

>    We do it for all our peach DNA. 

I dont have the original question but you may want to try a CTAB
extraction under high salt conditions.  The polysaccharides
precipitate with the CTAB and the DNA is then precipitated with
isopropanol.  The protocol is as follows.  I dont have the original
reference though, sorry.

CTAB Plant DNA Extraction

Buffer:
	Stock Solutions		Amount		[Final]
	1M Tris pH 7.5		10ml		100mM
	CTAB			1g		1%
	5M NaCl			14ml		0.7M
	0.5M EDTA		2ml		10mM
	BME			1ml 		1%
	dH2O			73ml 

Freeze and grind plant material in liquid N to a fine powder and then:
		
1)  Transfer 300-500mg powder to 10ml buffer (preheated to 65 deg) and
incubate 30-60 min. at 60 deg.
2)  Remove sample and allow to return to rm temp.
3)  Add 5ml CHCl3:Isoamyl (24:1) and invert gently for 5 min.
4)  Spin 10 min @ rm temp.
5)  Transfer aqueous phase to a new tube with 7 ml isopropanol.
6)  Mix quickly by inversion several times until a clot forms. Remove
clot with a glass hook.
7)  Rinse in 1-2 ml 76% EtOH, 0.2M NaOAc 20 min.
8)  Rinse in 1 ml 76% EtOH, 10mM NH4OAc and immediately transfer to
400µl TE.  DNA should slide easily off hook. (may take gentle rocking
overnite @ 4deg to completely redissolve)

There was also a protocol in Biotechniques that involved precipitating
polysaccharides out of solution with .35 vol EtOH.  It was in Vol. 17,
I think.

Good luck.

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523 Northwestern ave.            Ph#:  515-294-8626
Ames, IA  50010                 http://www.public.iastate.edu/~ippm
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