hello,I am constructing a Cas9-gRNA library using lentiV2 plasmid. My linearized vector has 13000bp, but the sgRNA fragment is only 89bp. If the molar ratio is 1:2 according to the instruction, the fragment only needs to be added a few ng, but I can't believe it. Has anyone done a similar experiment?
Kristin M. Adams
This is correct. The MW weight of a 13kb plasmid is much more than an 89bp plasmid.
0 votes
0 thanks
Michael J. Benedik
Yes, the mass ratio would be about 150 to 1 in order to get roughly equilmolar free ends.
As to whether that is the right ratio to use may depend on whether both ends have the same sticky end or are different, and whether or not all ends are phosphorylated. The optimal ratio might be different depending upon those.
0 votes
0 thanks
- Badges
- Science topic
More Lemon Li's questions See All
Similar questions and discussions