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digestion of gemone DNA

digestion of gemone DNA - Microbiology Forum

digestion of gemone DNA - Discuss Microbiology Science and Protocols here. Post questions on the study of viruses, fungi, parasites and bacteria here. Microbiology Forum.


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  #1  
Old 10-18-2004, 05:23 AM
grace_tsoi2@hotmail.com
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Default digestion of gemone DNA



I am trying to digestion the genome DNA of my strain,but find that it doesn't work with the enzyme Sau3A I for many hours.My guess is there are stuff along with the genome DNA which prevents Sau3A I from working.And my question is how and what exactly makes it fail to work.Any suggestions?Thank you!

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Old 10-18-2004, 01:31 PM
Tom Knight
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Default digestion of genomic DNA


[Only registered users see links. ] writes:

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.

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  #3  
Old 10-21-2004, 10:17 AM
Duncan Clark
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Default digestion of genomic DNA

Historians believe that in newspost
<[Only registered users see links. ].edu> on Mon, 18 Oct 2004, Tom
Knight <[Only registered users see links. ].edu> penned the following literary masterpiece:

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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