Sep 27, 2023
Preparation of Competent Cells (10β E. coli Strain)
- 1National University of Singapore
Protocol Citation: NUS iGEM 2023. Preparation of Competent Cells (10β E. coli Strain). protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7p5w9gwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 27, 2023
Last Modified: September 28, 2023
Protocol Integer ID: 88455
Keywords: Competent Cells, Transformation
Abstract
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.
Materials
- NEB 10-beta Competent E.coli (High Efficiency) - 6x0.2 mlNew England BiolabsCatalog #C3019I
- LB Media
- MgCl2 Solution
- CaCl2 Solution
- 100% Glycerol Solution
Safety warnings
- Proper lab PPE must be worn at all times.
- Thermal gloves shall be worn when handling cell stock from the -80°C fridge.
Cell Culture from Cell Stock
Cell Culture from Cell Stock
Prepare a Falcon tube with 5 mL of LB media.
Prepare an ice box.
Take out a tube of Sample cell stock from the -80 °C fridge and put it into the ice box.
In the biosafety cabinet (BSC), use an inoculation loop to inoculate some competent cells into the Falcon tube with 5 mL of LB media.
Incubate the cells in an incubator at 37 °C for Overnight
Refresh Cell Culture
Refresh Cell Culture
Prepare a new Falcon tube and add 10 mL of LB media into the tube.
Add 100 µL of the pre-cultured cells into this new Falcon tube to refresh the cells.
Incubate the cells at 37 °C for 02:00:00 .
2h
Pre-Cell Washing
Pre-Cell Washing
Pre-cool the centrifuge machine to 4 °C .
Take out the Falcon tube from the incubator, ensuring that the optical density (OD) of the cultured cells is 0.6OD to 0.8OD.
Place the Falcon tube in ice for 00:30:00 .
30m
Prepare 10 mL of 0.1M MgCl2 and 10 mL of 0.1M CaCl2, put both tubes in ice for 00:30:00 .
30m
Cell Washing
Cell Washing
Centrifuge the Falcon tube with cultured cells in the pre-cooled centrifuge machine at 5000 rpm for 00:05:00 . The temperature in the centrifuge machine must be kept at 4 °C the whole time in the "Cell Washing" section.
5m
Discard the supernatant and keep the cell pellet.
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.
Centrifuge the Falcon tube again at 5000 rpm for 00:05:00 .
5m
Discard the supernatant and keep the cell pellet.
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.
Place the Falcon tube in ice for 01:00:00 .
1h
Centrifuge the Falcon tube with the cells at 5000 rpm for 00:05:00 .
5m
Discard the supernatant and keep the cell pellet.
Storage
Storage
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.
Add 250 µL of glycerol-CaCl2 solution into the Falcon tube with the cell pellet and resuspend the cell pellet.
Split the cells in the Falcon tube into 5 new Eppendorf tubes with each tube containing 50 µL of cells.
The competent cells can be stored in a -80°C fridge or used immediately.