Lysing difficult "Bugs"

Koen De Smet kds30
Tue Feb 20 09:06:31 EST 1996


styler at HPB.HWC.CA (Shaun Tyler) wrote:
>
>I work with a wide range of bacteria (literally from A to Z) and have=20
>frequently had problems with lysing them for DNA extraction.  As most o=
>f=20
>my work has focused on PCR applications anything that doesn=92t lyse wi=
>th=20
>lysozyme or prot. K I have simply vortexed with glass beads in order to=
>=20
>crack them open.  However this also shears the hell out of the DNA and =
>it=20
>is not suitable applications which require high MW DNA.  I would be ver=
>y=20
>interested in hearing about ANY method others have used to achieve lysi=
>s=20
>from bacteria that don=92t lyse by standard methods.
>
>Shaun Tyler
>Head, DNA Core Facility
>Bureau of Microbiology
>Laboratory Centre for Disease Control
>Health Canada
>
>Ph #: (613) 941-6441
>FAX #:  (613) 957-1358
>
>E-mail:  styler at hpb.hwc.ca

As you probably know, mycobacteria are hard to lyse because they have an extra 
thick and waxy cell wall. I have lysed mycobacteria by spinning them down, 
suspending them in chlorophorm, vortex for 30 sec, add equal volume of water, spin 
and use aquaous phase in a PCR reaction.
To extract DNA for Southerns and so, we use a method based on SDS, CTAB and 
chlorophorm. Let me know if you are interested.
Succesful lysis depends on the age of the culture. It works best when they are 
still in log phase.

Good Luck,

Koen De Smet





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