DH5 α (containing pSABEn plasmid) Escherichia coli|360501 |BNCC

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Escherichia coli DH5α（pSABEn）

Subculture procedure	① Prepare 1 sterile water tube and 2 90mm resistant plates; ② After disinfecting the surface of the ampoule tube, open it in a safety cabinet, burn the top with an alcohol lamp, quickly drip sterile water to break it, and then use tweezers to crush it; ③ Draw 0.5mL of sterile water and transfer it into a freeze-drying tube. After sufficient dissolution, apply the solution onto resistant plates at a rate of 200 μ L per Agar plate; ④ Place the resistant Agar plate under the specified conditions for cultivation for 18-24 hours and observe it (sealing and wrapping the Agar plate is prohibited); ⑤ Select a single colony and inoculate it into 50mL of liquid LB resistant medium. Incubate overnight at 37 ℃ on a shaker; ⑥ According to experimental requirements, extract an appropriate amount of bacterial solution for plasmid extraction;
Growth conditions	37 ℃; 18-24h; aerobic
Storage conditions	2-8 ℃
Safety level	1
Sharing mode	Public welfare sharing

Escherichia coli DH5α(pSABEn)

Storage conditions: 2~8 ℃

No. 360501

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 receipt, please contact the customer service within 24 hours. If it is overdue, it is deemed that the product are well. The freeze dried culture shall be used up once and shall not be retained. Please operate in strict accordance with this instruction, otherwise the replacement of bacteria are not be available in case of aberrant growth and loss of viability.

Host bacteria:BNCC353719 DH5α

Plasmid:BNCC361033 pSABEn (containing kanamycin resistance gene).

Medium:LB resistant agar medium yeast extract 5.0g, peptone 10.0g, NaCl 10.0g, agar 15.0g (liquid medium not included), distilled water 1.0L, pH 7.0. Sterilize at 121°C for 15min. After sterilization, kanamycin was added at a concentration of 50mcg/ml when the medium was cooled to 40-50°C.

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 Open the ampoule 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 medium into a freeze dried ampoule, fully dissolve and transfer the solution to the test tube of host bacteria solution , mix evenly;

④ Draw partial solution from the test tube and spread it over the plate at 200ul/plate;

⑤ Put all the test tubes and plates under the above specified conditions for cultivation. The plates are not allowed to seal by wrapping. After 18-24 hours, the culture medium is obvious turbid and the growth of plate colonies or moss occurs.

Plasmid purification:

1. Pick a single colony and inoculate it into 50ml liquid LB resistance medium, shake it for cultivation at 37 ℃ overnight;

2. According to the requirements, draw appropriate amount of bacterial liquid for plasmid extraction.

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