Aug 06, 2024
Obtaining Competent Cells
- Miquel Estévez-Gay1
- 1Universitat de Girona
Protocol Citation: Miquel Estévez-Gay 2024. Obtaining Competent Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk86z1l5r/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: August 06, 2024
Last Modified: August 06, 2024
Protocol Integer ID: 104801
Abstract
Obtencion of 100ml competent E.coli aliquots that must be stored in -80C
Guidelines
Competent cells are fragile. All work must be done under a laminar flux cabin and in aseptic conditions.
Incubate 10ml of E.coli BL21(DE3) or DH5α o/n without plasmid (from –80°C stock). To do that, add 10 mL of sterile LB media in a 50ml sterile falcon tube. With the use of a yellow micropipette, scratch the surface of the frozen E.coli from the -80°C freezer and toss it into the Falcon containing the media. This must be done fast, and the E.coli must not be outside the freezer more than 1 or 2 minutes (Use the termoblock from the -20°C to keep the E.coli stock frozen).
Incubate Overnight 40 rpm, 37°C Rocker Mixer
Re-inoculate in a new 10 mL LB. Incubate until absorbance is 0.6-0.7 OD550 02:00:00 .
2h
Centrifuge 4000 rpm, 4°C, 00:10:00
10m
Under the Laminar flux Cabin, throw the supernatant and resuspend in the same volume of CaCl2 100 millimolar (mM) cold and sterile. Do not use vortex!
Incubate in ice 00:30:00 .
30m
Centrifuge again 4000 rpm, 4°C, 00:10:00
10m
Resuspend in 1/10th of the previous volume of cold and sterile CaCl2 and 15% glycerol. Aliquot them in 100 µL (using 1.5ml centrifuge tubes).
These aliquots can be used right away or can be stored in the -80°C freezer.