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Transferring mamallian cell colonies for selection


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#1 J85

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Posted 20 October 2009 - 09:04 AM

First post here, so thanks to anyone who can help me out.

I'm doing a neomycin selection on transfected human cells. After initially plating the cells post-transfection and allowing them to develop into colonies, I'm looking to transfer individual colonies to a 24-well plate. Any suggestions on how to maximize the efficiency of this transfer?

I've attempted this previously using bits of filter paper, but ended up with large quantities of paper debris in each well, and next to no cell growth. Is there a better method (or product) that can be used to ensure that the cells are actually transferred to each well?

Jon

#2 Dr Teeth

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Posted 20 October 2009 - 10:15 AM

First post here, so thanks to anyone who can help me out.

I'm doing a neomycin selection on transfected human cells. After initially plating the cells post-transfection and allowing them to develop into colonies, I'm looking to transfer individual colonies to a 24-well plate. Any suggestions on how to maximize the efficiency of this transfer?

I've attempted this previously using bits of filter paper, but ended up with large quantities of paper debris in each well, and next to no cell growth. Is there a better method (or product) that can be used to ensure that the cells are actually transferred to each well?

Jon



Try sterile cloning discs (from Scienceware, I think). Basically, you soak them in trypsin, place them over the colonies you want to transfer, allow a few minutes for trypsinization, and then transfer the disc to a 24 well with selective media. Other people use cloning cylinders, but I find these to be messy and more difficult to use.

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#3 J85

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Posted 20 October 2009 - 11:39 AM

First post here, so thanks to anyone who can help me out.

I'm doing a neomycin selection on transfected human cells. After initially plating the cells post-transfection and allowing them to develop into colonies, I'm looking to transfer individual colonies to a 24-well plate. Any suggestions on how to maximize the efficiency of this transfer?

I've attempted this previously using bits of filter paper, but ended up with large quantities of paper debris in each well, and next to no cell growth. Is there a better method (or product) that can be used to ensure that the cells are actually transferred to each well?

Jon



Try sterile cloning discs (from Scienceware, I think). Basically, you soak them in trypsin, place them over the colonies you want to transfer, allow a few minutes for trypsinization, and then transfer the disc to a 24 well with selective media. Other people use cloning cylinders, but I find these to be messy and more difficult to use.



Great suggestion, thanks. I'll look into ordering some.

Edited by J85, 20 October 2009 - 11:47 AM.





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