[Microbiology] Re: isolation of bacteria

Duncan Clark via microbio%40net.bio.net (by blackhole from abuse.plus.com)
Mon Sep 22 08:15:13 EST 2008


Historians believe that in newspost 
<mailman.82.1221933057.29717.microbio from net.bio.net> on Fri, 19 Sep 2008, 
RUTH SIELE <ruthsiele from yahoo.com> penned the following literary 
masterpiece:
>I am trying to isolate the bacteria from brackish water. I incubated the brackish water for three weeks. When I centrifuge the culture after three
>weeks, I couldn't get any pellets to extract the DNA. I streak them in LB plates but it will be culture dependent.

Incubate how?

What makes you think the bugs there will actually grow on LB? Some might 
but it's probably way off the optimum for most.

A chemical analysis of the water will give some insights to it's make 
up.

Failing that, at least try simply plating on brackish water agar plates 
i.e. filter the water and autoclave with agar.

> 
>Thus, I am looking for your suggestions if there is other means by which i can extract the DNA or if there is anything I have to do so as to get
>pellets from the culture

Take 10-100litres of water direct from the brackish water source. Simply 
centrifuge or concentrate by ultra filtration. Lyse cells from whatever 
pellet you get (possibly using one of the soil isolation kits from 
Qiagen or similar) and phi29 amplify whatever DNA is there using a 
Genomphi kit from GE Healthcare. If you can get 1-10ng of DNA to start 
with you will at least get 4-5ug back after amplification.

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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