JAM-A CRISPR/Cas9 KO Plasmid (h)

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

CRISPR/Cas9-mediated knockdown of hJAM1.
To knock down the F11R gene, HuH7, HepG2, and SK-CO15 cells were transfected with a mixture of JAM-A CRIPSR/Cas9 knockout and corresponding JAM-A HDR plasmids (Santa Cruz Biotechnology, Inc., Dallas, TX). Plasmid transfections were performed with UltraCruz Transfection Reagent (HuH7 and SK-CO15) or Lipofectamine 3000 (HepG2) in accordance with the protocols provided by the manufacturers (Santa Cruz Biotechnology, Inc., and Thermo Fisher Scientific, Inc., respectively). Efficiency of transfection was monitored by detection of the expression of vector-encoded GFP and RFP by fluorescence microscopy. Transfected cells were selected with medium containing puromycin (5 µg/ml for HuH7 and SK-CO15 cells and 2 µg/ml for HepG2 cells). Expanded pools of cells resistant to puromycin were maintained in the corresponding selective medium. After three or four passages, they were further enriched for cells expressing RFP by bulk sorting on a FACS Aria II (BD Biosciences, San Jose, CA) equipped with 488-, 405-, 561-, and 633-nm lasers and a 100-mm nozzle at a sheath pressure of 20 lb/in2. First, live cells were gated by using forward scatter area versus side scatter area. Three sets of gates that included side scatter height versus side scatter width, forward scatter height versus forward scatter width, and forward scatter width versus side scatter area were then used to exclude doublets. Cells were collected in 15-ml tubes with the corresponding growth medium. The same sorter was used to deposit single cells into 96-well plates for clonal selection. The clones obtained were expanded and assayed for hJAM1 expression. The knockdown of this protein was confirmed by flow cytometry and Western blot analyses. For flow cytometry, live cells (n = 2 × 106) were stained with FITC-conjugated anti-human CD321 (F11R) antibodies (BioLegend, San Diego, CA) as recommended by the manufacturer and analyzed with a BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA). Unstained cells and cells stained with FITC-conjugated isotype control antibodies (BioLegend) were used as fluorescent labeling controls.

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Manufacturer protocol

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

Download manufacturer protocol

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