pCW-Cas9

Protocol tips

Upstream tips	Protocol tips	Downstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate

Upstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate

Publication protocol

Vectors. For the construction of lentivirus vector NT-GFP, the NTCP gene obtained from Origene (Rockville, MD) was transferred into lentivirus vector pLVX-IRES-ZsGreen (Clontech, Mountain View, Ca). For the construction of pLX-SG1, a NdeI–AgeI fragment in pLX-AAVS1 sgRNA27 was replaced with a NdeI–AgeI fragment carrying two BsmBI/Esp3L restriction sites for cloning of guide RNA sequences with DNA oligomers (Supplementary Table S1). The nucleotide sequence of the NdeI–AgeI fragment in pLX-SG1 is shown in Supplementary Figure S1. sgRNA selection was based on algorithms developed by the Zhang group (crispr.mit.edu).

Virus and infections. HBV was concentrated 100-fold from the culture medium of HepAD38 cells with 6% polyethylene glycol and resuspended in serum-free Dulbecco's modified Eagle medium (DMEM)/F12 medium. Infection of HepG2 cells occurred in the presence of 4% polyethylene glycol in complete serum-free medium (DMEM/F12, pyruvate, nonessential amino acids, and penicillin/streptomycin) containing 2% dimethyl sulfoxide (DMSO) with an estimated 50 genome equivalents per cell. Medium was replaced with complete medium containing 10% fetal calf serum and 2% DMSO 24 hours after infection. Cultures were incubated for 5–10 days.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing CRISPR Human - Repression ENII-CP/X using pCW-Cas9 from Addgene.

Manufacturer protocol

Download the product protocol from Addgene for pCW-Cas9 below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing CRISPR Human - Repression ENII-CP/X using pCW-Cas9 from Addgene. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.