hCas9

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.	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.

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.
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

Generation of EGFP-2A-cas9, i20-gRNA, and i23-gRNA adenoviruses. EGFP-2A-cas9, i20-gRNA, and i23-gRNA cassettes were subcloned into pShuttle-CMV vector (Clontech, Mountain View, CA) and recombinant adenovirus genomic DNA were generated using the AdEasy-1 Adenovirus system (Agilent Technologies, La Jolla, CA) according to the manufacturer's instructions. The adenoviral particles were packaged and amplified in AD293 cells and purified by cesium chloride gradient ultracentrifugation followed by dialysis in storage buffer (10 mmol/l Tris-HCl pH 8.0, 2 mmol/l MgCl2, 4% sucrose). The titers of the adenovirus preparations were quantified by measuring the OD260.29

Cell culture and transfection. C2C12 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and electroporated with Neon Transfection System (Invitrogen, Carlsbad, CA). Briefly, 1 × 105 cells were electroporated with 0.25 µg cas9 and 0.125 µg i20-gRNA and 0.125 µg i23-gRNA plasmids. The electroporation conditions were 1,650 V, 10 ms, 3 pulses. Primary mdx myoblasts were isolated from the hind-limb skeletal muscles of mdx mice of 6 weeks old by digestion with collagenase type IA (Sigma-Aldrich, St Louis, MO), and cultured in DMEM/F-12 supplemented with 20% FBS. mdx myoblasts at 50–60% confluence were infected with EGFP-2A-cas9, i20-gRNA, and i23-gRNA adenoviruses at 100 multiplicity of infection. After 48 hours, C2C12 and mdx myoblasts were collected for the following genomic DNA analysis. Electroporated C2C12 cells were cultured in differentiation medium. mdx myotubes were harvested to analyze the dystrophin expression by RT-PCR after 3 days in differentiation medium.

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Manufacturer protocol

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