pSpCas9(BB)-2A-Puro (PX459)

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

The gRNA sequences for GLT25D1 exon 2 (forward: 5′-CACCGGAAGAGTTTGTACCATTCCG-3′, reverse: 5′-AAACCGGAATGGTACAAACTCTTC-3′), for GLT25D2 exon 3 (forward: 5′-CACCGCCATGTGATGAAACTACGAC-3′, reverse: 5′-AAACGTCGTAGTTTC-ATCACATGGC-3′), and for the control construct (forward: 5′-CACCGGAAGAGTTTGTACCTTTCC G-3′, reverse: 5′-AAACCGGAAAGGTACAAACTCTTC-3′) were ligated into the BbsI sites of pSpCas9(BB) (gift from Dr. Feng Zhang, Addgene plasmid no. 48139). Osteosarcoma cells were transfected using the AMAXA nucleofector kit (Lonza, Basel, Switzerland) according to the manufacturer protocol. The cells were selected for positive transfection with 0.5 μg/ml of puromycin (Santa Cruz Biotechnology Inc., Dallas, TX). Single clones were isolated and analyzed for mutations in the targeted genes using the Surveyor nuclease assay (Integrated DNA Technologies, Coralville, IA) (47) and the primers 5′-GGAGAAGTGTCCTGTCCAGGGATAC-3′ and 5′-ACAGGGAACGGCTTGGGCAAAGGTC-3′ for GLT25D1 exon 2 and 5′-CCCTGATGAAATTGGACCAAAGC-3′ and 5′-TGCCTTTCTTAAAAAGTGGGGG-3′ for GLT25D2 exon 3. Mutations were confirmed by Sanger sequencing (Microsynth).

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Discussion

Papers

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Manufacturer protocol

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

Download manufacturer protocol

Videos

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