Hello.
I'm using CRISPR technology for the first time. The kit I'm using is GeneArt® CRISPR Nuclease Vector with OFP Reporter Kit (Catalog number: A21174, Invitrogen).
My question is how much time should I wait from transfection (by Lipofectamine 2000) until being safe to use FACS for OFP enrichment of my cells (HEK).
Also, if I want to introduce puromycin (~600bp) resistance after the DS break, what kind of plasmid should I use for the homologous recombination repair?
Thank you!
Hi Dennis,
I'm not sure what exactly you plan to do, and I did not use this specific kit, but in any case, such reporters usually start to express very fast (after 3-4 hours you may already observe fluorescence). I would suggest to FACS-sort your cells 24-48 hrs after transfection.
Cheers,
Lech
Hello, Lech.
Thank you for your answer!
I was just wondering how much time would the plasmid remain in my cells after transfection (and thus, how much time I would have after transfecting to procede with the fluorescent sorting). I will use this window of 24-48 hours then.
@Zhi Liu,
I'm using the same kit but transfection efficiency is quite low. Say total 10 signals from a well (24-well format). My cell line is ovarian cancer cell line and have quite good growing ability. Would it okay for you to share transfection protocol? Thank you.
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