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help - stable transfection clone pick up - (Apr/08/2004 )

hi,
Since I am going to establish stable transfection,and now I have got many clones in plates. But how can I pick out those clones? I have tried sevral ways but not succeed.
I hope someone could suggest me some efficient methods to pick up the monoclones.
Thanks!

-geness-

Hello Geness,

There are many ways of picking clones

You can use a cloning cylinder (such as from Fisher)
To isolate incubated colonies, insert cloning cylinder around the cell colony and attach by sterile grease. Add trypsin to the cylinder and incubate until cells detach from the dish. Collect cells by a Pasteur pipet and transfer to separate vessel.

Or simply, without using cylinder, you can add trypsin to a well isolated cell clone and collect cells by pipetting.

Also have a look at this link http://anatomy.med.unsw.edu.au/cblonly/methods/mh10.htm

Good luck.

ZW

-zhongw90-

Thank you very much! I will have a try.
Good luck to you,too.

-geness-

Alternatively, you could seed them VERY sparse in a 100mm dish. After two-three weeks, you will be able to see single colonies, which you can pick using a Gilson. Simply put your tip in the colony and transfer to fresh media in a 24-well dish. I've tried that and found it easier then using these rings (they fall off very easily, so be carefull)

sprag

-Sprag-

You can also count your cells, dilute them at 0.5cell/100Ál of medium and plate 100Ál per well in a 96-wells plate. You should then have 1 cell every 2 wells wich assure you to have monoclonal population.

-Simonsays-

hybirdize

-antib-

The methods suggested by Simonsays and Sprag are feasible for me, and

I have tried them. The method of hybirdizing is new to me, would you

mind explain it in detail, antib?

Thank you!

-geness-

I have a question for Sprag and zhongw90.

Sprag, when you say "put tip in colony", do you use trypsin? or just scratch the cells?

zhongw90, how to remove the trypsin after detaching the cells? I doubt if centrifugation is appropriate since there are so few cells.

thank you!

-mystery-

I don't use any trypsin,... just suck them up with the tip using your Gilson..

-Sprag-

QUOTE
how to remove the trypsin after detaching the cells? I doubt if centrifugation is appropriate since there are so few cells.


You don't need to remove the trace trypsin.

-zhongw90-