Liquid Culture Amplification of V2 Human GeCKO library
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Andrew Manon
Dec 21, 2015, 1:35:37 PM12/21/15
to Genome Engineering using CRISPR/Cas Systems
Hi all,
Liquid culture amplification can theoretically introduce plasmid representation bias, as a previous post in this group suggests:
"Anybody could tell me why the GeCKO library should not be amplified in liquid cultures?"
Beyond the theory, however, does anyone have any experience with this method? I have difficulty seeing why any individual plasmid in the Human GeCKO V2 library would confer a selective advantage/disadvantage to the E. coli since 1) both the gRNA and Cas9 protein are driven by a eukaryotic promoter and 2) each plasmid varies only by 20bp.
Your insights are appreciated!
Rocamar
Dec 21, 2015, 4:39:31 PM12/21/15
to Andrew Manon, Genome Engineering using CRISPR/Cas Systems
I ll give it a try:-).dont know if the marker is amp:-)
In liquid culture, the beta lactamase that inactivated the ampicillin is secreted...and after prolonged cell growth in liquid, the selection pressure on the plasmid is weaken...
On solid plates...the beta lactamase diffuse within the agar and it appears this favours plasmid stability...
See bitesizebio ..they recall about ampicillin few things young researchers are not aware:-)
Didier fesquet
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Neville Sanjana
Dec 21, 2015, 6:56:38 PM12/21/15
to Rocamar, Andrew Manon, Genome Engineering using CRISPR/Cas Systems
Hi Didier and Andrew,
I haven't tried liquid amplification (mostly because of what I've heard from others) but some recent pooled CRISPR screen papers (e.g. Wang et al., Science, 2015) mention liquid culture.
If either of you try it, I'd be curious to hear what your experiences are. (It certainly would make things easier.) Please do report back if you try it and do sequencing on the result.
Best,
Neville
Nicky Thompson
Sep 15, 2016, 5:18:00 AM9/15/16
to Genome Engineering using CRISPR/Cas Systems, roca...@gmail.com, every...@gmail.com, nsan...@mit.edu
It has been tested: see this paper:
pragya sidhwani
Oct 6, 2022, 12:49:25 AM10/6/22
to Genome Engineering using CRISPR/Cas Systems
Hi all, did anyone end up trying this?
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