pET-28a - Basic Information

Recommended Medium	Yeast extract: 5.0g, Peptone: 10.0g, NaCl: 10.0g, pH: 7.0 (25℃)
Growth Conditions	LB + antibiotics, 37℃
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 1 min; ② Mixing: Mix 200μL competent cells with 5-10μL plasmid DNA, and place on ice for 30 min; ③ Heat shock: Place at 42℃ for 90 s; ④ Membrane pore contraction: Ice bath for 2 min; ⑤ Recovery culture: Add 800μL liquid Recommended Medium to each tube, and culture on a shaker at 37℃ for 1 h (150 r/min); ⑥ Screening culture: Spread an appropriate volume (100μL) of recovered cells onto LB plates with the corresponding antibiotic. Leave the plates upright for 30 min (the agar surface must be thoroughly dry), then invert and culture for 12-16 h until colonies appear; ⑦ Extraction: Pick single monoclonal colonies into liquid Recommended Medium with the corresponding antibiotic, culture with shaking for 12-16 h, and extract the plasmid as needed for the experiment.
Reference Use	pET-28a is a T7 promoter-based prokaryotic expression vector for E. coli, carrying N-terminal/C-terminal 6×His tags and kanamycin resistance, suitable for IPTG-induced high-efficiency expression and purification of recombinant proteins.
Sharing Method	Public welfare sharing

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