LB + 50mcg/ml ampicillin: yeast extract 5.0g, peptone 10.0g,NaCl 10.0g, distilled water 1.0L,pH 7.0. Sterilization at 121 ℃ for 15min. After sterilization, ampicillin was added when the culture medium was cooled to 40-50 ℃ with a concentration of 50mcg/ml.
After receiving the plasmid dry powder, please add 20 μl of sterile water to the bottom of the tube, dissolve the plasmid, and let it stand at room temperature for 1min;
2. Mixed (adsorption plasmid):
200 μl competent cells + plasmid DNA 5~10 & micro; L mixed evenly and placed on ice for 30min;
3. Heat shock introduction:
Let stand at 42 ℃ for 90s;
4. Shrink film hole:
Ice bath for 2min;
5. Repair culture:
Add 800 & micro to each tube; L LB liquid medium, cultured at 37 ℃ for 1h 150 r/min;
6. Screening and cultivation:
The appropriate volume (100 & micro; L) resuscitated cells are coated on the LB plate with corresponding resistance, and placed in the plate for 30min (after the agar surface must be dried),
Inverted culture for 12-16h, colonies appeared.
Pick the monoclonal colonies into the corresponding resistant LB liquid medium, shake culture for 12-16h, according to the test needs to extract the plasmid.