pCTCon2|357429 |BNCC

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pCTCon2

Subculture procedure	1. After receiving the plasmid powder, please centrifuge at 5000rpm for 1 minute, then add 20 μ l sterile water to dissolve the plasmid, and let it stand at Room temperature for 1 minute; 2. Remove the corresponding sensory state from the -80 ° C refrigerator, thaw it on an ice box, and mark it properly; 3. Take 2 μ l of plasmid and add it to 100 μ l of the competent state, then ice bath for 30 minutes; 4. Heat shock at 42 ° C for 90 seconds, then ice bath for 2 minutes; 5. Add 900 μ l of LB liquid medium without resistance and shake at 180rpm for 45 minutes; 6. Centrifuge at 6000rpm for 5 minutes, leaving only 100 μ l of supernatant to mix and precipitate the bacterial cells; 7. Add the mixed bacterial solution onto the corresponding resistant LB Agar plate, pour in an appropriate amount of glass beads, and spread the liquid evenly; 8. Cultivate the Agar plate in the forward direction for 1 hour, and then invert it for 12h-16 hours; 9. Select monoclonal colonies and culture them in LB liquid medium with corresponding resistance, shake for 12h-16h, and extract plasmids according to experimental needs. Attention: If the transformation Agar plate grows too much the next day, please dilute the plasmid proportionally before transformation.
Growth conditions	37℃； 18-24h; aerobic
Storage conditions	2-8 ℃
Safety level	1
Sharing mode	Public welfare sharing

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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