quick colony hybridisation method?

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Mon Apr 3 10:37:41 EST 2000


In article <38E81974.415FE403 at hort.cri.nz>,
Kimberley Snowden <ksnowden at hort.cri.nz> wrote:
> Does anyone out there have a reliable quick method for doing colony
> hybs? When I'm doing library screening I do everything properly - ie
> plate the bugs on a membrane then replica to other membranes which I
> then probe, and I have no problems with this method. However sometimes
> if I'm cloning something tricky I like to do colony hybs to identify
> potential positives. But I don't want to go through all the plating and
> replica'ing - instead I'd like to just lift the colonies that I get on a
> transformation plate, and probe this. The problem I have when I try this
> though is that sometimes either the colonies don't stick to the
> membrane, or the colonies come totally off the plate onto the membrane
> and are no longer on the plate. And often both will happen on the same
> plate. Does anyone have a reliable method for getting good sticking of
> all colonies to the membrane, while also leaving plenty of the colony on
> the plate so I can go back and pick positives later?
>
> thanks
> Kim
>
>

I like to try to get them all to stick on the membrane. You can put the
plate back in the incubator overnight to grow them back. There will be
enough bugs left on the plate to seed a new colony. You'll probably get
some satellites, but you can work around them.

I also usually chill the plate in the refig. before lifting to firm up
the agar. I still get some colonies that don't lift well, but if you are
just screening recombinant clones rather than  library screening, it's no
big deal. You usually only need one good one, anyway.

--
_______________________________________________
Nick Theodorakis
nicholas_theodorakis at urmc.rochester.edu


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