I an recently trying to transfect 293T cells with a plasmid which should utilize as a homolog sequence for HDR in genome editing via CRISPR/Cas9. since this is not working for me for quite a while now, I was thinking of linear plasmid insertion. once I cut the plasmid, do I need to dephosphorilate it as well in order to avoid it from circulating again?
Thanks!!
Hello,
The donor template DNA does not need to be linearized. It is not
recommended to linearize it as this will increase the random integration but If You decide to do this I would suggest to linearize your vector, ideally with two restriction enzymes.
1. Seems like you want to use CRISPR/Cas9 and NHEJ (instead of HDR) repair pathway to knock-in your insert, it should be better to use linear DNA. As suggested, linear DNA is more efficient for stable transformation in a cell transfection experiment (see 'transient transfection' vs. 'stable transfection' at link below): ( https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/transfection-basics/guidelines-for-plasmid-dna-transfection.html.)
2. I am not sure about re-ligation in cell, but as Jaroslaw already suggested, it is a good idea to use REs which can linearize your vector with two incomparable ends to prevent relegation (if any).
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