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

Generation of single guide RNA encoding plasmids
Single guide (sg)RNAs targeting exon 1 of the murine β2m gene or the IAb beta chain gene were designed using the online sgRNA design tool available at https://crispr.mit.edu/. In order to maximize specificity, guide sequences with high scores for on-target activity and at least 3 base pair mismatches to any predicted off-targets in the genome were selected. The corresponding sense and antisense DNA oligomers (obtained from Sigma) are shown in S1 Table. Annealed oligomers were cloned downstream of the U6 promoter of PX458 (Addgene, Plasmid 48138, Middlesex, UK) as described earlier (Ran, 2013). A plasmid map of PX458 is depicted in S1 Fig.

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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.

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Videos

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