Hi everyone I am new to CRISPR- CAS, and have designed the gRNA for a gene. i did the transfection in fibroblast cells. After that I used Genomic Cleavage Detection (GCD) Kit and got the few bands (uncut and cut products) in 3% agarose gel. Although the cut product was faint but I guess it could be because of lower transfection efficiency. Can anyone please tell me who to screen the cells in a population having transfected material?
Hi Ankita,
Transfected cells can be enriched by antibiotics selection or FACS sorting.
Hie ankita
You can perform FACS cell sorting, if the vector you used have any fluorescence tag or you can do antibiotic selection that too if your vector carry that resistance. After which, you can do single cell dilution and then expand the cells to perform different experiments such as sequencing, off-target analysis, western blotting to check expression etc
If the vector you have used do not carry above mentioned tags, in that case you can try co-transfection with another vector having these tags.
Hi
we know Fluorescent tags are enormous, if a Fluorescent tag like GFP (with others) inserted into genome maybe leading instability up in Genome .
its just a theory
BEst
Hi! Actually to answer you, I need to ask some questions:
Did you first optimize your transfection protocol? and what do you transfect (gRNA + Cas9 mRNA or RNP complexes)?
In my opinion, it's essential before going for genome editing. Many parameters can influence transfection: cell density, ratio reagent:RNA or reagent:RNP, duration of transfection, etc
Did you use controls with the GCD kit?
Looking forward,
Best,
Sandra
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