17 replies to this topic

[geness]

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!

[zhongw90]

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.u...ethods/mh10.htm

Good luck.

ZW

[geness]

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

[Sprag]

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

[Simonsays]

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

[antib]

hybirdize

[geness]

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!

[mystery]

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!

[Sprag]

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

Edited by Sprag, 19 November 2004 - 07:41 AM.

[zhongw90]

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.

[WHR]

geness, on Sep 13 2004, 05:21 AM, said:

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!

I had not tried this method. In short, you put a small piece of filter paper containing trypsin on the colony, wait for a few minutes, and then lift it up. There should be some cells on the paper.

[WHR]

If your cells form compact colonies, scraping the colonies using a pipette tip is easy, as Sprag suggested.

Edited by WHR, 19 February 2009 - 10:27 PM.

[scolix]

try to pick using a small pipette tip. If needed use a microscope while picking the cells.

[rkay447]

I recommend you isolate single cells.  This is the only way to be absolutely certain that your resulting cell line is a monoclonal population.  The problem with isolating by ring or hybridizing (I have tried this and it does work) is you may be getting a mix of different cells.  Who knows if the colony you see arised from one cell, two, three... The last thing you want to do is experiments and work just to find out your line is a mix.  When I made my lines I would trypsinize colonies using the rings and transfer each into a 6 well.  Immediately I would use a glass micropipette (just melt and pull a normal one over a bunsen burner) and using a microscope, isolate individual cells from each colony and transfer to a 96-well.

[farzana]

I am a new starter in making of a stable cell line from MCF 7 cell. Could any one guide me in this regard. How do i start?