Can I use NEB mutagenesis -- substitution method to clone sgRNA into the vector?
I felt it's much easier. Just design the primers containing the sgRNA bp, conduct PCR and do a ligation. And most of the clones will be sgRNA positive, no need to worry about failing to cut a restriction site and having negative clones.
Dooyoung Lee
Hi Suipwksiow,
I've tried this method in the past and worked well, although personally I prefer restriction enzyme cloning because most of the genome-editing vectors are relatively large. It is easier for me to prepare microgram quantity of completely digested vector at once and ligate different sgDNA duplexes rather than to do long PCR for every construct.
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