Stable Transfection Clone Pickup?

Stable Transfection Clone Pickup?

Postby medicbio on Fri Jul 11, 2014 4:14 pm

I'm trying to establish a stable transfection, and currently I have lots of clones on my plates. How do I pick up those clones and isolate them from the other colonies?
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Re: Stable Transfection Clone Pickup?

Postby bioforum on Fri Jul 11, 2014 4:38 pm

There are several options here:
-One is to use a cloning cylinder (available from VWR, Fisher, etc), which attaches with sterile grease around the cell colony. Then you add trypsin to the cylinder and incubate until the cells detach from the plate. Collect the cells with a pipet and transfer to your new container.
-If a clone is well-isolated, you can just add trypsin directly to it and collect with a pipet.
-If you can seed your cells extremely sparsely in a 100 mm plate, after 2-3 weeks you should see single colonies that you can just pick up with a micropipettor. Some of my colleagues find this easier than using the rings.
-Yet another option is to count your cells and then dilute them to 0.5 cell/100 microliters of media. You then plate them at 100 microliters per well in a 96 well plate. The dilution ensures that there is only one cell for every two wells which ensures that each well has a monoclonal population.
-No one I know has tried this method, but another way is to put a filter paper with some trypsin soaked into it on the colony and wait a few minutes. After waiting, lift away the filter and there should be some cells on it that can be used for your next step.
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