"Gently Resuspend"
Brett Beitzel
Subject:"Gently Resuspend" Date: 5/23/96
After struggling to "gently resuspend" a pellet of DH5alpha that I am trying
to make competent, I got to wondering how other people "gently resuspend"
bacterial cell pellets. I used a 10ml pipet, but this left some small pieces
that didn't want to get any smaller. I've tried "swirling" the cells around
in fresh buffer - didn't work too well; vortexing isn't exactly "gentle" (by
the way, how gentle do you have to be?) Anybody have other tricks for
resuspending pellets?
Just Curious,
Brett
brett_...@msmtp.idde.saci.org
Cancer Therapy and Research Center
San Antonio, TX
Richard Daines
Brett Beitzel wrote:
>
> Subject:"Gently Resuspend" Date: 5/23/96
>
I never pipet the cells but flick the tube to make a gentle swirl. This
takes time to fully resuspend the pellet. Try not to centrifuge too
hard. Never use a pipetman. If you have to pipet to break up clumps,
then use a wide bore.
--
Richard J. Daines
Mystic, CT
Sailesh Surapureddi
Hello there!
I have used a 10mL pipet for 'Gentle resupension'. All I did was rinse
the pipet 2 to 3 times in cold buffer and then for the cells, they were
initially swirlled and then pipetted up and down 10-12 times using
powerpette on ice. All the cells were suspended properly and I got
reasonably good yeilds. So I guess this much of harsh treatment, the cells
would be able to take without crying out loud.
Cheers
Sailesh.
Ned Mantei
In article <n1379255...@msmtp.idde.saci.org>,
brett_...@MSMTP.FS.SACI.ORG ("Brett Beitzel") wrote:
> Subject:"Gently Resuspend" Date: 5/23/96
>
> After struggling to "gently resuspend" a pellet of DH5alpha that I am trying
> to make competent, I got to wondering how other people "gently resuspend"
> bacterial cell pellets.
One possibility is to take a fine glass rod, or a Pasteur pipet with the
end melted shut, and stir a bit. This brings the pellet away from the
bottom of the tube and it is then easier to resuspend.
--
Ned Mantei
Dept. of Neurobiology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland
man...@neuro.biol.ethz.ch Fax: +41-1-633-1046
Marcel Hoffmann
Brett Beitzel wrote:
>
> Subject:"Gently Resuspend" Date: 5/23/96
>
> After struggling to "gently resuspend" a pellet of DH5alpha that I am trying
> to make competent, I got to wondering how other people "gently resuspend"
> that didn't want to get any smaller. I've tried "swirling" the cells around
> in fresh buffer - didn't work too well; vortexing isn't exactly "gentle" (by
> the way, how gentle do you have to be?) Anybody have other tricks for
> resuspending pellets?
>
> Just Curious,
Hai Brett,
When I have harvested my bacteria a little bit of medium
(LB) is left on the bacteria. Just in this little bit of
medium I resuspend my bacteria by vortexing. Then I add my
icecold buffer to this suspension of bacteria. I use a
protocol to make competent cells that just takes
resuspending the cells only once after harvesting them.
Good luck
Dr. Marcel Hoffmann
Marcel Hoffmann
Marcel Hoffmann
Chris Boyd
: After struggling to "gently resuspend" a pellet of DH5alpha that I am trying
: to make competent, I got to wondering how other people "gently resuspend"
: bacterial cell pellets. I used a 10ml pipet, but this left some small pieces
: that didn't want to get any smaller. I've tried "swirling" the cells around
: in fresh buffer - didn't work too well; vortexing isn't exactly "gentle" (by
: the way, how gentle do you have to be?) Anybody have other tricks for
: resuspending pellets?
I find the best tool for this job is the disposable plastic Pastette
(wide bore tip for large samples, narrow bore tip for small ones).
Just pump up and down a few times with it until the pellet is fully
dispersed, remembering to keep the vessel on ice. It also helps not to
spin the cells down too hard: 6K for 5 min in a GSA-type rotor should
be hunky dory. "Gentleness is everywhere, don't play the game of
time."
Best wishes,
--
Chris Boyd | from, | MRC Human Genetics Unit
chr...@hgu.mrc.ac.uk | not | Western General Hospital
http://www.hgu.mrc.ac.uk/~chrisb | for | Edinburgh EH4 2XU, SCOTLAND