pLV2-EF1a-mKeima-Puro-Basic Information

Recommended Medium	Yeast extract: 5.0g, Peptone: 10.0g, NaCl: 10.0g, pH: 7.0 (25℃)
Growth Conditions	37℃； 18-24 hours； Aerobic；
Storage Conditions	2-8℃
Procedure	① Dissolution: Add 20μL sterile water to dissolve the plasmid dry powder, 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 transformation: Place at 42℃ for 90s； ④ Membrane pore contraction: Ice bath for 2min； ⑤ Recovery culture: Add 800μl liquid Recommended Medium to each tube, culture at 37℃ with shaking for 1h (150 r/min)； ⑥ Selection culture: Spread an appropriate volume (100 μl) of recovered cells onto LB plates with the corresponding antibiotic, keep plates upright for 30min (ensure agar surface is completely dry before use), 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, and extract plasmids as required for the experiment
Reference Application	pLV2-EF1a-mKeima-Puro is a lentiviral expression vector that uses the EF1a promoter to drive expression of mKeima (an acid-sensitive fluorescent protein) for monitoring autophagy or tracking acidic organelles, and carries a Puromycin resistance marker for selection of stable cell lines.
Sharing Method	Public Welfare Sharing