sgRNA Library construction using LentiCRISPRv2 - Help!!!

[Ahmed Elwakeel]

Hi everyone, 

I am currently trying to implement Tim Wang protocol (CSH 2016) to construct a lentiviral library to do pooled CRISPR screen. 

Could you please let me know how I could calculate the amount of insert (DNA oligos) and vector (digested LentiCRISPRv2) required for Gibson Assembly cloning of 452 gRNAs?

Thanks in advance. 

Ahmed Elwakeel

University of Cambridge       

[Eric Shi]

maintaining a molar ratio of 3:1 (insert: vector) should be a good starting point. also make sure you have enough colony coverage. 452 sgRNA is not a lot, you should be able to get the library easily.