pHAGE-3×FLAG-MCS-Puro - Basic Information

Recommended Medium	Yeast extract: 5.0g, Peptone: 10.0g, NaCl: 10.0g, pH: 7.0 (25°C)
Growth Conditions	37°C; 18-24 hours; Aerobic;
Storage Conditions	2-8°C
Procedure	① Dissolution: Add 20μL sterile water to the plasmid dry powder for dissolution, and let stand at room temperature for 1 min; ② Mixing: Mix 200μl competent cells with 5-10μl 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: Place on ice for 2 min; ⑤ Recovery culture: Add 800μl liquid Recommended Medium to each tube, and culture at 37°C on a shaker 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 (ensure the agar surface is completely dry first), then invert and culture for 12-16 h until colonies appear; ⑦ Extraction: Pick single colonies into LB liquid Recommended Medium with the corresponding antibiotic, culture with shaking for 12-16 h, and extract plasmids as needed for the experiment.
Reference Use	pHAGE-3×FLAG-MCS-Puro is a lentiviral expression vector that drives expression of the target gene inserted into the multiple cloning site (MCS) with an N-terminal 3×FLAG tag under the CMV promoter, and enables stable cell line selection through puromycin resistance.
Sharing Method	Public Welfare Sharing