digestion of genomic DNA

Duncan Clark blackhole at abuse.plus.com
Thu Oct 21 05:17:16 EST 2004


Historians believe that in newspost 
<vuyr7nwgnkw.fsf at soggy-fibers.csail.mit.edu> on Mon, 18 Oct 2004, Tom 
Knight <tk at csail.mit.edu> penned the following literary masterpiece:
>grace_tsoi2 at hotmail.com writes:
>>   I am trying to digest the genomic DNA of my strain, but find that
>>   it doesn't work with the enzyme Sau3A I after many hours.  My guess is
>>   there are stuff along with the genomic DNA which prevents Sau3A I
>>   from working.  My question is how and what exactly makes it fail
>>   to work.
>
>You don't say what your strain is, but there are many bacteria that
>have methylases which add methyl groups selectively to A or C residues
>in genomic DNA.  My organism, for example, methylates the C in the
>sequence GATC, and is completely resistant to Sau3AI digestion, as
>well as resistant to BamHI digestion, due to the internal GATC site in
>the GGATCC recognition sequence for BamHI.  So although your DNA may
>be inhibiting the reaction, it may also be that the GATC sites are
>5-methyl C methylated.

Simple to check.

Add a plasmid or a.n.other bit of DNA to your xsomal digest. Run 
appropriate controls.

If the plasmid gets digested on its own with no xsomal present then the 
RE and buffer are OK.

If the plasmid gets digested in with the xsomal then there are no 
inhibitors in the xsomal prep and xsomal methylation is the probable 
cause.

If the plasmid does not digest in with the xsomal then the xsomal prep 
has inhibitors still present i.e. phenol, SDS, proteinase K etc etc.

Duncan
-- 
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.



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