SMS1 CRISPR/Cas9 KO Plasmid (m)

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 SMS1 and SMS2 KO Cells Using CRISPR/Cas9 KO System
Sphingomyelin synthase 1 (Sgms1) and 2 (Sgms2) CRISPR/Cas9 knock-out plasmids (sc-431565 and sc-428888) were purchased from Santa Cruz Biotechnology, Inc. We followed the manufacturer's protocol. Briefly, C2C12 myoblasts were seeded in a 6-well plate in 3 ml of antibiotic-free 10% FBS DMEM, a day prior to transfection. When cells reached ∼50% confluence, the plasmid DNA solution composed of 2 μg of CRISPR/Cas9 KO plasmid and 2 μg of the corresponding HDR plasmid (sc-431565-HDR and sc-428888-HDR) in 15 μl of UltraCruz transfection reagent (sc395739) was added dropwise to each well. The cells were incubated and monitored for 24–72 h. Successful co-transfection of the CRISPR/Cas9 KO plasmid and HDR plasmid was confirmed by detection of red fluorescent protein under a fluorescence microscope, and the cells were selected with media containing puromycin to generate the KO cell line. After 1 week of incubation with puromycin, the gene expression was assessed by RT-qPCR following RNA isolation and cDNA synthesis. Primer sequences used herein correspond to the gRNA sequences in the CRISPR/Cas9 KO plasmids.

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Manufacturer protocol

Download the product protocol from Santa Cruz Biotechnology for SMS1 CRISPR/Cas9 KO Plasmid (m) below.

Download manufacturer protocol

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