I am using guide RNA and crispr RNA so dont have any selection markers to screen the transfected cells.
If you plan on nucleofecting your cells, I would recommend using a Cas9-GFP RNA or protein in order to sort your cells post transfection. The screening process will be extremely laborious if you do not use a selection strategy.
Typically a minimum of 1x10^6 - 3x10^6 cells for nucleofection, followed by cell sorting based on the Cas9-GFP 24hrs post-transfection. This way, one can determine how many cells go into a single well, I typically perform single-cell sorting so that subsequent colonies are clonal GFP+, giving me the highest probability for targeting events or indels upon screening.
Hope this helps
Thank you so much. Your suggestion is indeed very helping. But is Cas9-GFP RNA commercially available?
Yes, both the plasmid and the synthetic sgRNA are available commercially. There are options for both the two plasmid approach and the single sgRNA-2A-CAS9-GFP. Sigma sells them as well as several other companies. The RNA can be purchased through GE and others as well. The synthetic option allows you to order guides without having to perform in vitro transcription.
Best of luck.
I have already got my guide RNAs through in vitro transcription and after your suggestion, I wrote to Sigma but they said they donot have any Cas9GFP RNA. If you do not mind then please give the catalog number and your mail id so that I can correspond with you directly. thanks
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