pT3-EF1a-MCS-3×Flag-P2A-EGFP-Basic Information

Recommended Medium	Yeast extract: 5.0g, Peptone: 10.0g, NaCl: 10.0g, pH: 7.0 (25℃)
Growth Conditions	37℃; 18-24h; Aerobic
Storage Conditions	2-8℃
Procedure	① Dissolution: Add 20μL sterile water to the plasmid dry powder to dissolve, let stand at room temperature for 1min; ② Mixing: Mix 200μl competent cells with 5-10μl plasmid DNA, place on ice for 30min; ③ Heat shock introduction: Let stand at 42℃ for 90s; ④ Membrane pore contraction: Ice bath for 2min; ⑤ Recovery culture: Add 800μl liquid Recommended Medium to each tube, culture on a shaker at 37℃ for 1h (150 r/min); ⑥ Screening culture: Spread an appropriate volume (100 μl) of recovered cells onto LB plates with the corresponding antibiotic, leave plates upright for 30min (ensure the agar surface is completely dry), then invert and culture for 12-16h until colonies appear; ⑦ Extraction: Pick single monoclonal colonies into liquid Recommended Medium with the corresponding antibiotic, culture with shaking for 12-16h, extract the plasmid according to experimental needs.
Intended Use	pT3-EF1a-MCS-3×Flag-P2A-EGFP is a plasmid vector used for protein expression in mammalian cells, utilizing the T3 and EF1a promoters to drive expression of the target gene fused with a 3×Flag tag, and co-expressing the EGFP reporter gene through the P2A self-cleaving peptide for tracking.
Sharing Method	Public Welfare Sharing