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Extrc. of DNA some G+ bacteria

Helgi Briem Magnusson hbriem at rhi.hi.is
Tue Aug 24 06:30:05 EST 1993


In <25cofj$jnm at ratatosk.uninett.no> viggo.lindahl at biotekn.nlh.no (Viggo Lindahl) writes:

>We are planning to extract DNA for PCR from extremely small (>0.4!m) gram
>positive soil bacteria. We know that these bacteria are very difficult to
>disrupt (we have tried high energy ultrasonic treatment for other
>purposes with no effect on the cells).

>I wonder if anybody have a good protocol to follow. I have been told that
>the use of SDS preferably should be omitted due to interference with the
>PCR, and also that methods not extracting RNA is preferable. (I don!t see
>why RNase treatment prior to ethanol precipitation can interfere with the
>PCR reaction).

I am currently working with an extremely tough Gram+ bacterium that
seems to resist most disruption methods like yours.  I am working
with tissue samples, so I use a slightly different method, but try
this:

Spin in a microfuge and remove supernatant

Resuspend in 10mM TrisCl (I use about 50ul, you may need more)

Add 25U Achromopeptidase (Sigma)

Incubate 1 hr 37C with shaking

Extract with phenol/chloroform

Resuspend in TrisCl

Clean with CTAB (see Ausubel or Sambrook for details)

EtOH precipitate, 70%EtOH wash and resuspend

Our bug, Renibacterium salmoninarum, is immune to all detergents
we have tried as well as most of the usual enzymes, ie ProteinaseK,
Lyozyme, Mutanolysin etc.  Achromopeptidase is the only enzyme
we have found that will open it.  Buy the purified version though,
the crude one didn't work for us.

Good Luck,
Helgi Briem
Inst.Exp.Path.
Keldur, Iceland



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