pGL3-U6-sgRNA-PGK-mCherry - Basic Information

Recommended Medium	Yeast extract: 5.0 g, Peptone: 10.0 g, NaCl: 10.0 g, pH: 7.0 (25°C)
Growth Conditions	37°C; 18-24 h; Aerobic
Storage Conditions	2-8°C
Procedure	① Dissolution: Add 20 μL of sterile water to the plasmid dry powder to dissolve it, and let stand at room temperature for 1 min; ② Mixing: Mix 200 μL of competent cells with 5-10 μL of plasmid DNA, and place on ice for 30 min; ③ Heat-shock introduction: Let stand at 42°C for 90 s; ④ Membrane pore contraction: Ice bath for 2 min; ⑤ Recovery culture: Add 800 μL of liquid Recommended Medium to each tube, and culture on a shaker at 37°C for 1 h (150 r/min); ⑥ Selection culture: Spread an appropriate volume (100 μL) of recovered cells onto LB plates with the corresponding antibiotic. Leave the plates upright for 30 min (make sure the agar surface is completely dry), then invert and culture for 12-16 h until colonies appear; ⑦ Extraction: Pick a single monoclonal colony into liquid Recommended Medium with the corresponding antibiotic on LB, culture with shaking for 12-16 h, and extract the plasmid according to experimental needs.
Intended Use	pGL3-U6-sgRNA-PGK-mCherry is an sgRNA expression vector for CRISPR gene editing in mammalian cells. It uses the U6 promoter to drive sgRNA transcription and the PGK promoter to drive mCherry red fluorescent protein as a reporter gene. It also carries a puromycin resistance marker for screening positive cells.
Sharing Method	Public welfare sharing