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

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	There is no need to perform a negative control golden-gate reaction (without insert) as it will always contain colonies so not a good indicator of cloning success.

Protocol tips
There is no need to perform a negative control golden-gate reaction (without insert) as it will always contain colonies so not a good indicator of cloning success.

Publication protocol

CRISPR/Cas9.
Ptgs2−/− SL4 cells were generated by CRISPR/Cas9-mediated genome engineering using the CRISPR design tool (www.genome-engineering.org). The genomic sequence of the Ptgs2 gene (5′-GCGGACTCCACGTGACGTAG-3′ and 5′-CAATAGTCTTAATGGCTTAC-3′) was targeted. Oligonucleotides corresponding to these guide sequences were cloned into the BbsI site of pSpCas9(BB)-2A-GFP (PX458) (Addgene), a bicistronic expression vector encoding both Cas9 and the single guide RNA. The expression vector construct was transiently transfected in SL4 cells, and then GFP-expressing cells were sorted by FACSAria (BD Biosciences).

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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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