Extrc. of DNA some G+ bacteria

Viggo Lindahl viggo.lindahl at biotekn.nlh.no
Tue Aug 24 04:53:55 EST 1993


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).

Bollet et al.,1991 (ref. below) mentions that treating the bacterial
pellet for 2 x 1 min at 900W in a microwave oven greatly improves the
yield of extracted DNA from G+ bacteria (among others). Does anybody have
any experience with this.

Obtaining our microbacteria for DNA extraction is rather expencive and
laborious, and an efficient extraction method would be appreciated by us.

Thank you from Viggo Lindahl

Ref: Bollet et al., 1991, Nucleic Acids Research 19, p. 1955



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