MYH9 CRISPR/Cas9 KO Plasmid (h)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
If transfecting more than one plasmid (i.e., CRISPR/Cas9 KO Plasmid with HDR Plasmid), mix Plasmid DNA at equivalent ratios.	Do not add antibiotics to the Plasmid Transfection Medium

Upstream tips
If transfecting more than one plasmid (i.e., CRISPR/Cas9 KO Plasmid with HDR Plasmid), mix Plasmid DNA at equivalent ratios.
Protocol tips
Do not add antibiotics to the Plasmid Transfection Medium

Publication protocol

Generation of Myh9 Knockout Cells Using CRISPR/Cas9 System
MYH9 clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) knockout plasmid (Santa Cruz Biotechnology, Dallas, TX, USA) consists of a pool of three plasmids, encoding three guide RNAs (gRNAs) to target different regions of Myh9 gene for maximum knockout efficiency. The gRNAs sequences targeted by Myh9 CRISPR plasmids are 5′-TGGTTCAAAGCCATTCTTGG-3′, 5′-ACCAGCCAGCCTTAAGGAGG-3′ and 5′-TATCTACTCAGAGGAGATCG-3′. Control CRISPR/Cas9 Plasmid does not recognize any DNA sequence used as a negative control (NC). Each plasmid encodes Cas9 nuclease and green fluorescent protein (GFP). PC12 cells were seeded in 6-well plates (at a density of 1 × 106 cells/well) the day before transfection. Cells were transfected with 2 μg of the plasmid using lipofectamine 3000®. After incubation of 72 h, GFP-positive cells were sorted and grown as single cell in 96-well plates using the BD FACSAria III cell sorter (BD Biosciences, San Jose, CA, USA). Colonies derived from single cells were expanded in media with 20% FBS. After 3 weeks, Myh9 knockout cells were screened by Western blotting using myosin IIA-specific antibodies.

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Manufacturer protocol

Download the product protocol from Santa Cruz Biotechnology for MYH9 CRISPR/Cas9 KO Plasmid (h) below.

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