pSpCas9(BB)-2A-GFP (PX458)

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

CRISPR-Cas9 sgRNA design and construction
The design of sgRNAs was carried out using the online program available from Zhang’s laboratory45. This program provides additional information about the quality of sgRNA by giving it a score value of up to 100 and additionally, the number and sites of potential off targets. Designed and processed sgRNAs to edit either B3GNT5 or B4GALNT1 (Supplemental Table S1) were cloned into pSpCas9(BB)-2A-GFP (addgene PX458) via BsbI restriction site using T4 DNA Ligase (Promega, Dübendorf, Switzerland).

IGROV1 and SKOV3 cells were transfected using ViaFect Transfection reagent (Promega, Dübendorf, Switzerland). A total of 4–5 × 105 cells were seeded into 6-well plates, cultured for 24 h and transfected with 2.5 μg of pSpCas9(BB)-2A-GFP. Cells were harvested after 72 h and subjected to single cell sorting.

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Reviews

Discussion

Papers

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Manufacturer protocol

Download the product protocol from Addgene for pSpCas9(BB)-2A-GFP (PX458) below.

Download manufacturer protocol

Videos

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