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

Genome engineering
Two protospacer-adjacent motifs (PAMs) 89 bp upstream and 30 bp downstream of the miR-322-binding site (miR-322bs) were identified using the CRISPR Design Tool (Ran et al., 2013). The single guide sequences 20 bp upstream of the PAMs were inserted into the sgRNA-expressing plasmid pSpCas9(BB)-2A-Puro(PX459)V2.0 to generate the PX459-Mek1UTR-5′-miR-322bs and the PX459-Mek1UTR-3′-miR-322bs targeting vector. The plasmid was a gift from Feng Zhang (Broad Institute of MIT and Harvard, Cambridge, MA, USA) (Addgene plasmid 62988). For transfection 5×105 ATDC5 cells were seeded in a 24-well culture plate and cultured in DMEM (Gibco) supplemented with 5% FCS at 37°C and 5% CO2. The next day, 500 ng of each targeting vector, 1.5 µl FuGENE (Promega) and 50 µl OptiMEM were added and incubated for 24 h. Transfection solution was replaced with selection medium containing 3 µg/ml puromycin (Sigma) and after 4 days cells were detached with trypsin/EDTA (Biochrom). A limited dilution was performed and a single cell was seeded into an individual well of a 96-well culture plate in the presence of medium without puromycin selection. Clones were expanded and isolated DNA was screened by PCR analysis for correct deletion of the miR-322 binding site. A 560 bp fragment was amplified for the wild-type and a 445 bp fragment for the mutated sequences. Primer sequences are provided in Fig. S3. These cells were then transfected with control or mimic oligonucleotides using the transfection protocol for PEC.

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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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