Need help with clonal isolation - (Apr/03/2012 )

Hi Everyone!

Really need your help and advice on this one. I'm generating a stable cell line with a neuroblastoma line. Most of the time these cells will grow in colonies but will be all spaced out from one another. I'm trying to isolate these colonies by pouring 1% Agar on them (37 degrees C of course), letting the agar polymerize and then use a separator to isolate, trypsinise and collect my colony. The problem is my cells almost immediately peel off the plate when I pour and get all mixed up in the Agar... so that method is out. Next I tried to isolate single cells to let them divide in separate wells - I scratch them off carefully and suck them up with a Gilson pipette and empty my pipette into a well. Problem with this method is I'm not seeing any cells in the wells (probably hurting my chances by transferring a single cell). I was wondering if there was a method where I could use a colony separator to extract whole colonies and increase the number of cells I put in a single well, without the Agar.

Need to find some way to keep the separator still while I add the trypsin and extract different colonies. Any thoughts?

Thanks for your help

The two common methods for this are to either do limiting dilution or to use cloning rings. Limiting dilution is performed by diluting the trasnfected cells out into 96 well plates such that you are only likely to get one cell per well. Cloning rings involves you seeding the cells out, selecting using an agent that kills untransfected cells, then allowing colonies to grow, then placing a cloning ring over the colony and trypsinising it so that you can lift only that colony out of the plate.