pacAd5-EF1a-MCS-IRES-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 h; Aerobic
Storage Conditions	2–8°C
Procedure	① Dissolution: Add 20 μL of sterile water to the plasmid dry powder to dissolve, and let 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 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 (the agar surface must be completely 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 required by the experiment.
Reference Use	pacAd5-EF1a-MCS-IRES-EGFP-Puro is presumed to be an expression vector based on the adenovirus (Ad5) backbone, using the EF1a promoter to drive expression of the target gene in the multiple cloning site (MCS), and linking EGFP (enhanced green fluorescent protein) and the Puromycin resistance gene through an IRES (internal ribosome entry site) element, enabling co-expression of the target gene, fluorescent tracing, and antibiotic selection.
Sharing Method	Public welfare sharing

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