TITLE: Preparation of Competent Cells (10β E. coli Strain)
AUTHORS: NUS iGEM

[DESCRIPTION]
2023 NUS-Singapore iGEM team followed this protocol to make competent cells that would be used for transformation.  

[GUIDELINES]
This protocol demonstrates the process of making 5 tubes of competent cells from a 5 mL cell culture. Generally, within our protocol, every 5 mL of cultured cells can be transformed into 5 tubes of competent cells, with each tube containing 50 µL of cells.


[STEPS]
SECTION: Cell Culture from Cell Stock
1. Prepare a Falcon tube with 5 mL of LB media.


SECTION: Cell Culture from Cell Stock
2. Prepare an ice box.

SECTION: Cell Culture from Cell Stock
3. Take out a tube of   cell stock from the -80 °C fridge and put it into the ice box.

SECTION: Refresh Cell Culture
6. Prepare a new Falcon tube and add 10 mL of LB media into the tube.

SECTION: Refresh Cell Culture
7. Add 100 µL of the pre-cultured cells into this new Falcon tube to refresh the cells.

SECTION: Refresh Cell Culture
8. Incubate the cells at 37 °C for 120 min.

SECTION: Storage
22. Prepare a 1 mL mixed solution composed of 20% glycerol and 80% 0.1M CaCl2 solution, and put it into the ice box to cool its temperature down. 
SECTION: Cell Culture from Cell Stock
5. Incubate the cells in an incubator at 37 °C for   

SECTION: Cell Culture from Cell Stock
4. In the biosafety cabinet (BSC), use an inoculation loop to inoculate some competent cells into the Falcon tube with 5 mL of LB media.

SECTION: Pre-Cell Washing
9. Pre-cool the centrifuge machine to 4 °C.

SECTION: Pre-Cell Washing
10. Take out the Falcon tube from the incubator, ensuring that the optical density (OD) of the cultured cells is 0.6OD to 0.8OD.
SECTION: Pre-Cell Washing
11. Place the Falcon tube in ice for 30 min.

SECTION: Pre-Cell Washing
12. Prepare 10 mL of 0.1M MgCl2 and 10 mL of 0.1M CaCl2, put both tubes in ice for 30 min.

SECTION: Cell Washing
13. Centrifuge the Falcon tube with cultured cells in the pre-cooled centrifuge machine at 5000 rpm for 5 min. The temperature in the centrifuge machine must be kept at 4 °C the whole time in the "Cell Washing" section.

SECTION: Cell Washing
14. Discard the supernatant and keep the cell pellet.
SECTION: Cell Washing
15. Add a small amount of MgCl2 solution prepared in the earlier step into the Falcon tube to resuspend the cell pellet. Then, pour the rest of the MgCl2 solution into the Falcon tube.
SECTION: Cell Washing
16. Centrifuge the Falcon tube again at 5000 rpm for 5 min.

SECTION: Cell Washing
17. Discard the supernatant and keep the cell pellet.
SECTION: Cell Washing
19. Place the Falcon tube in ice for 60 min.

SECTION: Cell Washing
20. Centrifuge the Falcon tube with the cells at 5000 rpm for 5 min.

SECTION: Cell Washing
21. Discard the supernatant and keep the cell pellet.
SECTION: Cell Washing
18. Add a small amount of CaCl2 solution prepared in the earlier step into the Falcon tube to resuspend the cell pellet. Then, pour the rest of the CaCl2 solution into the Falcon tube.
SECTION: Storage
23. Add 250 µL of glycerol-CaCl2 solution into the Falcon tube with the cell pellet and resuspend the cell pellet. 

SECTION: Storage
24. Split the cells in the Falcon tube into 5 new Eppendorf tubes with each tube containing 50 µL of cells.

SECTION: Storage
25. The competent cells can be stored in a -80°C fridge or used immediately.