Scaling transfections for smaller libraries

I am trying to plan for a small CRISPR screen (~125 genes including negative controls, 5 guides/gene, so only about 6-700 unique guides in lentiguide-puro) in a tricky cell.

This is kind of a general question specifically re: transfections to produce lentivirus, but what scale would you start at to ensure that your viral prep adequately represents your library as well? When trying to scale down from the Joung et al Nat Protocols paper, I think it seems like a single 10cm plate (for example) would produce more than enough virus for a small library like this, so long as you titer an aliquot for MOI/infectivity? Do you just assume that as long as the NGS of your cloned library has sufficient representation of all guides that this means they will be evenly represented in your viral prep as well, even if you have poor transfection efficiency? Is there some math to ballpark the representation of each guide (ie ug of vector DNA<-->mols/copies divided by library size or something like that)?


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Lemon Lamiaabout 3 years ago

this paper is a straightforward guide to the math behind libraries, specifically protein engineering libraries. Although it is not specific to your situation, I hope it helps a bit.

https://doi.org/10.1016/j.bioeng.2004.11.002

Fuchsia Kelpieabout 3 years ago

For the Thermo T225/T75/T25's, I scale linearly based on the area of the flask. For instance, to scale T225 down to T75, I use a third of all reagents in the T225 transfection (i.e. 15mL of media instead of 45mL).

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