'Cracking'

D. KIM dkim at NMSU.Edu
Fri Jun 11 19:18:59 EST 1999


I believe this "direct lysis" protocol comes up semiannually in
BioTechniques.  I have had some success with"

1.  Scoop up the colony with a yellow tip.

2.  Suspend in a small volume of TE, RNAse A, lysozyme, 10% glycerol (use
concentrations that make sense to you.  Maybe omit lysozyme.)

3.  Transfer into the well of an agarose gel.  If this is done quickly,
the cells will not have lysed, and will leave the pipet intact.  If you
wait too long, the chromosomal DNA will make pipetting almost impossible.

4.  When all wells are filled, ovelay them with an equal (to the
bacterial suspension) volume of NaOH/SDS with loading buffer (to make it
sink and to give it color).  You may see the bacterial suspension "clear"
in the well after a minute.

5.  Run the gel.

I don't know if the lysozyme or RNAse A make a difference at all, butI
have the mixes ready already, so it doesn't inconvenience me to use them.
Chromosomal DNA seems to stay in the well (perhaps trapped by the bactrial
"shells").  Plasmid DNA appears to be in supercoiled configuration.  RNA
runs quickly down the gel, but may exhaust the ethidium bromide in the
lane as it travels.  This technique may therefore give more consistent
migration patterns if the gel is stained after the run.

I don't have a citation, either.

Daniel Kim
dkim at nmsu.edu

Bernard P. Murray, PhD <bpmurray*STUFFER*@socrates.ucsf.edu> wrote:

: I've occasionally used the protocol in Sambrook et al. (page 1.32).

: One of my co-workers says he once used a really nice protocol
: which involved an agarose gel containing detergent and the
: bacteria were loaded (whole) into this.  Alas, he has neither the
: protocol nor the reference for this method.

:    Bernard

: -- 
: Bernard P. Murray, PhD
: Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



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