pSpCas9(BB)-2A-Puro (PX459) V2.0

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-mediated genome editing and RNA interference
RNA interference (RNAi) and genome editing of murine B16-F1 and NIH3T3 cells was done as before21. The NIH3T3 F2/3 KO clones #9 and #46 described to lack FMNL3 but to still express in-frame deleted variants of FMNL2 at very low dose21, were now additionally treated with CRISPR/Cas9 plasmid pSpCas9(BB)-2A-Puro (Addgene plasmid ID:48139) encoding FMNL2 target sequence 5′-GTCATACTGCCGCAGTAGCC-3′, now mediating gene disruption within exon 2. Upon isolation of new individual colonies, their expansion and exploration by Western Blotting, novel clones F2/3 KO #9/10 and F2/3 KO #46/20 completely devoid of FMNL3 and residual FMNL2 protein expression were selected and characterized further in the present study.

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Discussion

Papers

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Manufacturer protocol

Download the product protocol from Addgene for pSpCas9(BB)-2A-Puro (PX459) V2.0 below.

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Videos

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