Escherichia coli DH5α(pEC-XK99E)|BNCC358941 |BNCC

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Escherichia coli DH5α(pEC-XK99E)

Culture medium	LB + 50mcg/ml kanamycin: yeast extract 5.0g, peptone 10.0g,NaCl 10.0g, distilled water 1.0L,pH 7.0. Sterilization at 121 ℃ for 15min. After sterilization, kanamycin was added when the culture medium was cooled to 40-50 ℃ at a concentration of 50mcg/ml.
Subculture procedure	(1) Prepare a test tube containing 5~10mL of liquid medium and 2 plates; (2) open it in the biosafety cabinet, heat the tip of ampoule in a flame, quickly drop sterile water to creak it, then break it with forceps; (3) draw 0.5mL of liquid culture medium into a freeze dried ampoule, fully rehydrate and transfer the solution to the liquid test tube, mix evenly; (4) inoculate a plate with 0.2mL of suspension liquid, repeat the step to obtain two plates; ⑤ Put all the liquid test tubes and plates under the above culture conditions for cultivation, and the strains can be used when they grow.
Growth conditions	37 ℃;18-24h; Aerobic
Storage conditions	2-8 ℃
application	pEC-XK99E plasmid extraction.
Sharing mode	Public welfare sharing

Escherichia coli DH5α(pEC-XK99E)

Storage conditions : 2~8 ℃

No. : 358941

Product format :freeze dried, 200ul

Validity : 6 years

Biosafety level : 1, handle in ultra-clean table or safety cabinet

Receiving notice: if any abnormality is found on the day of receiving goods, please contact customer service within 24 hours. if the goods are overdue, the goods will be deemed as good. Dry powder shall not be retained once used up. Please activate in strict accordance with this instruction, otherwise the replacement service will not be provided if the strain is abnormal and inactivated.

Host bacteria:BNCC353719 DH5α.

Plasmid:BNCC357431 pEC-XK99E (containing kanamycin resistance gene).

Medium:LB resistant agar medium yeast extract 5.0g, peptone 10.0g,NaCl 10.0g, agar 15.0g (not included in liquid medium), distilled water 1.0 L,pH 7.0. Sterilization at 121 ℃ for 15min. After sterilization, cooling and cooling, add kanamycin to make the final content of kanamycin in the culture medium 50μg/mL.

Growth conditions:37 ℃ aerobic 18-24h.

Recovery steps:

1. Prepare 1 sterile liquid test tube (including 5~10mL of culture medium) and 2 pieces of 90mm plate;

2. After disinfecting the surface of the ampoule tube, open it in the safety cabinet, burn the top with an alcohol lamp, quickly drop sterile water to break it, and then break it with tweezers;

3. Suck 0.5mL of liquid culture medium and put it into the freeze-drying tube, fully dissolve it and return it to the liquid test tube again and mix well.

4. Draw the mixed solution from the test tube liquid to apply the flat plate,200μL/piece;

5. Put all the test tubes and plates under the above specified conditions for cultivation. It is forbidden to seal and wind the plates. After 18-24 hours, observe the obvious turbidity of the culture medium and the growth of plate colonies or moss.

6. If the strain has not grown, it should continue to be cultured for 72h until a single colony or moss grows.

Plasmid purification:

1. pick a single colony and inoculate it into 50mL liquid LB resistance medium, and culture at 37 ℃ overnight;

2. according to the experimental requirements, absorb appropriate amount of bacterial liquid for plasmid extraction.

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