Protocolo paraq fabricar celulas ultracompetentes

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  • Publicado : 25 de abril de 2010
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Ultra-Competent E. coli Cells

Preparation:
• Prepare SOB media and Transformation Buffer.
• Sterilize 6, 250 mL centrifuge bottles; 100 mL graduated cylinder.
• Pre-cool the rotor,buffer, and sterile plastic ware (above plus sterile 25 mL pipet, pipet tips, and 600 μL tubes) in the cold room.
• Set up 600 μL tubes in 1 mL pipet tip racks on ice during spins and incubationson the final day.

Day 1:
1. Take 100 μL competent cells and inoculate a 10 mL SOB culture with selection. Grow overnight at 37°C.

Day 2:
2. Inoculate each of 6, 500 mL flaskscontaining 200 mL SOB (with selection) with 1 mL of the overnight culture. Grow at 18°C, shaking at 200-250 rpm until
OD600 = 0.6. (This may take up to 3 days.)

Final Day:
First Spin -
3.Transfer contents of each flask to a centrifuge bottle and balance them.
4. Incubate bottles on ice for 10 minutes.
5. Spin at 4100 rpm (2500 X G) and 2°C for 10 minutes in rotor coded 28, SorvallRC 5C Plus centrifuge.
6. Place bottles on ice and take to cold room.

First Resuspension – IN COLD ROOM
7. Pour off supernatant into flasks and invert bottles on paper towels.
8.Gently resuspend pellets (by swirling) in 67 mL (1/3 original volume) Transformation Buffer.
9. Incubate on ice 10 minutes.

Second Spin
10. Spin down cells exactly as before.
11. Place bottleson ice and take to cold room.

Final Resuspension – IN COLD ROOM
12. Pour off supernatant into flasks as before.
13. Resuspend pellets in 17 mL (1/12 original volume) Transformation Buffer.14. Add 1.19 mL DMSO (595 μL twice, 7% final concentration), swirling gently.
15. Incubate on ice 10 minutes.
16. Dispense 210 μL cells into pre-cooled 600 μL tubes.
17. Freeze with liquidN, and store immediately in -80°C.

SOB Medium
2% Bacto-tryptone
0.5% yeast extract
10 mM NaCl (0.6 g/L)
2.5 mM KCl (0.18 g/L)
10 mM MgCl2 (MgCl2-6H2O 2.03 g/L)...
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