pT7-6×His-SacB - 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 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℃ 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 on a shaker at 37 ℃ for 1 h（150 r/min）； ⑥ Screening culture: Spread an appropriate volume (100 μl) of recovered cells onto LB plates with the corresponding antibiotic resistance, leave the plates upright for 30 min (the agar surface must be 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 resistance, culture with shaking for 12-16 h, and extract the plasmid according to experimental needs.
Reference Application	pT7-6 His-SacB is an E. coli expression plasmid based on the T7 promoter, with the SacB gene as its core element. The SacB gene encodes levansucrase, which produces a strong lethal effect on E. coli in sucrose-containing Recommended Medium. Therefore, this plasmid is often used as a counter-selectable marker in experimental scenarios such as gene editing or mutant screening where positive clones need to be eliminated.
Sharing Method	Public Welfare Sharing

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