pLV3-CMV-3×FLAG-MCS-EF1a-EGFP-Puro - 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 hours; 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 it 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 (the agar surface must be dry), then invert and culture for 12-16 h until colonies appear; ⑦ Extraction: Pick single monoclonal colonies into liquid LB Recommended Medium with the corresponding antibiotic, culture with shaking for 12-16 h, and extract the plasmid as needed for the experiment.
Reference Application	pLV3-CMV-3×FLAG-MCS-EF1a-EGFP-Puro is a lentiviral vector that uses the CMV promoter to drive fusion expression of the 3×FLAG tag and the target gene in the multiple cloning site (MCS), while an independent EF1a promoter drives co-expression of EGFP and Puromycin, enabling protein tagging, fluorescence tracing, and stable cell line selection.
Sharing Method	Public welfare sharing