making glycerol stock of CRISPR Library

[Minoo karimi]

Hi all,

I am making a custom sgRNA library that contains 100,000 oligos. I would like to keep several glycerol stocks from this library but I am about confused about the procedure considering we do not incubate the cells in liquid culture overnight.

1-How to determine the number of cells per each glycerol vial to maintain the diversity of the library? 

2-can I store some parts of the cells after scaping the colonies off from the lb agar plates before plasmid extraction? 

Thanks,

Minoo

[Julia Joung]

Hi Minoo,

I never make glycerol stocks when I'm cloning or amplifying sgRNA libraries. We minimize the colony growth time to 12-14h to avoid difference in growth rates between colonies expressing different sgRNAs that could increase the skew of the library.

After scraping off the bacteria, you can spin down and store the pellet at -20C. I would recommend maxiprepping all of the bacteria together to maintain screening coverage.

Best,

Julia

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[Minoo karimi]

Thanks Julia for the comment. 

Minoo