probs digesting plant genomic

[Chi-Kuang Wen]

BOTPG at LUFF.LATROBE.EDU.AU (GRANT,Patricia) wrote:
>
>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
Hi, Trish:

   Have you tried to use other restriction enzymes to digest 
your DNA preparations?  My experience is that XhoI does not
cut plant DNA very well, or almost does not cut.  I have never
used PvuII for this purpose.  Several enzymes I have used are
EcoRI, BamHI, HindIII, PstI and XhoI and PstI and XhoI almost
do not cut the DNA and the resulting DNA still remains at the
high molecular weight position.  What people think about this
problem is DNA-methylation which occurs at GC and plant genomic
DNA is always highly methylated.  To test if your DNA is 
methylated, you may include plasmid DNA with your plant DNA and 
subject this mixture for restriction enzyme digestion, if the 
plasmid DNA is cut and the plant DNA is not cut, then we might
be able to say that the methylated DNA is resistant to some 
types of restriction enzymes.  
  By the way, many restrictions are methylation-sensitive and 
level and position of methylation may affect the digestion of 
the DNA.

regards,

Chi-Kuang