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 genome engineering
CRISPR/Cas9 genome engineering was performed as described (Ran et al. 2013). To delete the MIR genomic sequence, preserving its 3′ end region, we used two short guide RNAs (sgRNAs) (see Supplemental Fig. S4A). After cotransfection of mouse ES cells with pSpCas9(BB)-2A-GFP vectors containing sgRNA1 and sgRNA2 and selection by FACS, we screened for MIR deletions by PCR on genomic DNA of single-cell-derived ES cells with a pair of primers flanking the MIR (Supplemental Fig. S4B) and by sequencing of some of the PCR products (Supplemental Fig. S4C). We obtained different deletion lengths (Supplemental Fig. S4D).

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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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