probs digesting plant genomic

[Kirrily O'Connor]

In article <4ejsk0$hod at lugb.latrobe.edu.au>, BOTPG at LUFF.LATROBE.EDU.AU 
says...
>
>
>I'm trying to digest genomic DNA from mature leaf tissue for eventual 
ligation 
>and use in inverse-PCR. I have been having problems trying to get the 
>stuff to digest with the restriction enzymes of choice (PvuII or XhoI). 
I've 
>tried several methods of genomic isolation including a quick and dirty 
>arabidopsis method (thought its not arab I'm working on), then CsCl 
gradient 
>then CTAB. None of these produced DNA which would cut with either of the 
>enzymes. Finally I have included Qiagen columns (anion exchange) as the 
final
>purification step and it now cuts when I use BamHI but won't with either 
of 
>the others. Does anyone have any suggestions....? 
>
>Thanks
>
>Trish

I'm really interested to know what plant you are working on.  Our lab has 
been having similar problems with digesting plant genomic DNA, mostly 
with rainforest plants.  We assume that the problem is with 
polysaccharides.  I have tried a number of ways of getting rid of these. 
 We use an extraction technique which I can give you the reference of if 
you like - I don't have it handy.  It's a modified CTAB extraction which 
separates the nuclei from the rest of the cytoplasmic gunk.  Although it 
produces DNA which we can actually pipette after the final precipitation 
(ie it usually has the consistency of honey or jelly with any other 
technique) it often will not restrict.  The only way that we can get it 
to restrict is to gel purify (from low melt agarose) the DNA.  Apart from 
being really tedious, I've found that my samples seem to degrade much 
faster after doing this.  

A couple of methods that I have tried which have not actually worked very 
well for me (but may do for you) are:

1. DNA double dissolution

To 500 microlitres of DNA (in TE or water) add 50 microlitres of 3M NaCl 
and 50 of beta-mercaptoethanol.  Put on ice for 15min then spin down.  
Any polysaccharides should precipitate as a clear jelly.  Transfer 
supernatant to a fresh tube and precipitate with 2 vols ethanol.

2.  Lithium chloride precipitation

Add an equal volume of 8M LiCl and let stand overnight at -20 degrees.  
Spin down for 15 min in a microfuge.  (another variation is to heat the 
sample at 60 degrees for an hour before adding the LiCl)

I'm not sure what you mean by a caesium chloride gradient method.  Is it 
the one that uses an ultra centrifuge and vast quantities of ethidium 
bromide (yuk!) ?  This method has worked for me but I prefer not to use 
it unless only doing one or two samples at a time because of its very low 
yield and the time and danger involved.

I would be really interested if anybody else has solutions to this 
problem ie how to effectively get rid of polysaccharides or even if it is 
polysaccharides that are causing the inhibition of restriction.

Kirrilee