Advice Needed: Using BbsI for Plasmid Cloning of Multiple gRNAs

[Salima Abu Rabe'a]

Hello, fellow researchers,

I'm working on a project cloning 11 guide RNAs into a Pxr003 plasmid using BbsI enzyme for cutting. How can I best approach this to ensure successful ligation of all guides? Any specific protocols or considerations I should be aware of?

Do you have any tips on optimizing gRNA selection and ensuring efficient digestion and ligation with BbsI? 

Appreciate your expertise!

Salima Abu Rabe'a, M.Sc. student

Prof. Gil Ast laboratory 

Departure of Human Molecular Genetics & Biochemistry

Sackler Faculty of Medicine

Tel Aviv University, Israel