pCMV-MCS-P2A-mCherry-3×NLS(repeat-opt)-Neo-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, and let stand at room temperature for 1min； ② Mixing: Mix 200μl competent cells with 5-10μl plasmid DNA, and 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, and culture on a shaker at 37℃ for 1h（150 r/min）； ⑥ Selection culture: Spread an appropriate volume (100 μl) of recovered cells on LB plates with the corresponding antibiotic. Leave the plates upright for 30min (the agar surface must be thoroughly dry), then invert and culture for 12-16h until colonies appear； ⑦ Extraction: Pick single monoclonal colonies into liquid LB Recommended Medium with the corresponding antibiotic, culture with shaking for 12-16h, and extract plasmids according to experimental needs.
Intended Use	pCMV-MCS-P2A-mCherry-3×NLS(repeat-opt)-Neo is a plasmid vector used for subcellular localization in mammalian cells. It uses the CMV promoter to drive target gene expression and co-expresses mCherry red fluorescent protein together with an optimized 3×NLS nuclear localization signal through the P2A self-cleaving peptide to trace the nucleus, while carrying neomycin (Neo/G418) resistance for stable cell line selection.
Sharing Method	Public welfare sharing

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