pIRES2-EGFP|BNCC354585 |BNCC

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pIRES2-EGFP

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 ℃ with a concentration of 50mcg/ml.
Subculture procedure	1. Dissolve: 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. 7. Extraction: 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.
Growth conditions	LB + kanamycin, 37 ℃ (clone strain DH5a,5308bp)
Storage conditions	2-8 ℃
Safety level	1
application	Protein expression
Sharing mode	Public welfare sharing

Plasmid instructions

Product format: freeze dried, 1.0-2.0ug
Valid period: 90 days
Storage temperature: -20℃
Shipping: at ambient temperature,within a week
Notes: The plasmids shall be transformed into competent cells, and then use after amplification and extraction. Before transformation, please contact technicians or visit the website for the information about the name and concentration of the antibiotics, competent cells and culture temperature.

Handling procedures:
1 dissolving: upon receipt of the freeze dried plasmid, add 20ul of sterile water to the bottom of the ampoule, dissolve the pellets, and sit at room temperature for 1min;
2 mixture (adsorption of plasmids ): mix 200ul competent cells and 5-10ul of plasmid DNA evenly and place it on ice for 30min;

3 heat shock: sit at 42 ℃ for 90s
4 shrink the membrane pore: ice bath for 2min
5 repair cultivation: add 800ul of LB liquid medium to each tube, culture at 37 ℃for 1 hour, 150 r / min;
6 screening cultivation: distribute appropriate (100ul) recovered cells to the LB plate with corresponding antibiotics, place the plate uprightly for 30min (the agar surface must be dried), sit the plate upside down for 12-16h, and colonies appear.
7 Extraction: pick the monoclonal colonies into the LB liquid medium with corresponding antibiotics, shake for 12-16h, and extract the plasmid according to the needs.

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