Recovering transformed cells from Soft Agar

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Amtekoth
Amtekoth's picture
Recovering transformed cells from Soft Agar

I've transformed human PBMCs with a virus and plated in soft agar (agarose, not low melt).

Colonies grew and I pulled plugs with pipet tips, plating the plugs in media in a 96 well plate. I had to pipet up and down a lot, since the plugs frequently stuck to the inside of the pipet tip.

I'm not sure if my cells were able to escape the agarose. Since I want to grow up transformed colonies, how do I get them out of the soft agar and growing happily?

Dumb side question: I know you can use agarase to recover nucleic acids from agarose. Would that work or would it kill my cells?

Thanks,

Ed

marcus muench
marcus muench's picture
I think if you break up the

I think if you break up the agarose enough that cell will find their own way out into the medium.

I'm not aware of any way to melt, even low-melting point agarose, and get viable cells out. I always use methylcellulose instead of agarose when I want to harvest colonies.

sjwilliams
sjwilliams's picture
You can recover both RNA, DNA

You can recover both RNA, DNA and protein from colonies growing in agarose by using the Tri-Reagent LS (MRC) using their protocol for extracting nucleic acids from cells/tissue with high lipid/glycoprotein content.  Juset ask the tech services.  It works very well for RNA and DNA extraction.  Protein is a bit tricky but high abundant protein is easy.

Good luck