Apr 03, 2019
Version 1
Making electro-competent E. coli cells and transformation of them V.1
- Jasper Koehorst1
- 1Wageningen University & Research
- IBISBATech. support email: [email protected]
Protocol Citation: Jasper Koehorst 2019. Making electro-competent E. coli cells and transformation of them. protocols.io https://dx.doi.org/10.17504/protocols.io.vzfe73n
Manuscript citation:
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: November 29, 2018
Last Modified: April 03, 2019
Protocol Integer ID: 18183
Keywords: E. coli, electro-competent, transformation, colicells electro, making electro, electroporation, competent cell, preparation, cell, electro, hours of preparation, culturing step
Abstract
This protocol describes a method to makeEscherichia colicells electro-competent. The method involves one overnight culturing step, followed by several hours of culturing during the day and 1.5 - 2 hours of preparation of electro-competent cells.
The protocol also describes how to do the transformation (electroporation) afterwards.
Guidelines
- The reason for resuspending the cells in 5 ml glycerol solution first before adding the remainder of the 50 ml is that it is easier to resuspend in a smaller volume than in a completely filled tube.
- Competent cells can be stored for 6 to 12 months at -80°C
- Work sterile during the whole procedure (next to bunsen burner or in flow cabinet)
- After transformation, the cells are spread onto a selective plate. To assure that single colonies appear, two plates can be used instead: one to spread 1/10 of the cells onto, and another one to spread 9/10 of the cells onto. If the transformation was very efficient, the single colonies will appear on the 1/10 plate and a lawn will appear on the 9/10 plate. In the case of a less efficient transformation, single colonies will appear on both plates or only on the 9/10 plate (and nothing on the 1/10 plate).
Materials
MATERIALS
GlycerolCatalog #G5516
conical tubes, 50ml
Electroporation System Gene Pulser XCellBio-Rad Laboratories
NanoDrop spectrophotometerThermo Fisher ScientificCatalog #ND-1000
Shaker incubator
Ice
Centrifuge
Thermomixer C or R EppendorfCatalog #5382000015 / Z605271
Liquid nitrogen
Liquid LB medium
Escherichia coli
This needs some work and investigation if we can specify a specific medium with the ingredient list there.. or we do it here
For making competent cells:
- 500 ml LB w/o salt (per liter: 10 g tryptone, 5 g yeast extract)
- 1 l ice-cold 10% v/v glycerol (per liter: 100 ml glycerol)
For transformation:
- SOC medium, 0.5 ml per transformation: (per liter: 20 g tryptone, 5 g yeast extract, 0.5 g NaCl, 0,186 g KCl – adjust to pH 7 with NaOH – autoclave – add 5 ml of sterile 2M MgCl2(which is 19 g MgCl2in 100 ml) and 4,5 ml of sterile 80% w/v glucose)
- Selective agar plates, depending on the plasmid that will be transformed into E. coli
For making competent cells:
- Centrifuge for disposable 50 ml tubes that can be cooled to 4°C
- Shaking incubator
- Spectrophotometer
For transformation:
- Thermomixer or water bath at 37°C
- Electroporation machine
For making competent cells:
- 1 sterile 50 ml tube for culturing (Corning mini bioreactor centrifuge tube 50 ml, #431720)
- 8 sterile 50 ml tubes (generic ones, not the ones for culturing)
- 20-30 sterile 1.5 ml eppendorf tubes that don’t pop open when submerged in N2(l)
- 1 sterile 1 L Erlenmeyer flask
For transformations:
- Sterile cooled electroporation cuvette with a 2 mm gap
- Sterile 1 ml syringe
- Sterile needle (BD Microlance; 21G 1.5”- Nr. 2; 0,8 x 40 mm; REF 304432; or a thicker needle)
Troubleshooting
Safety warnings
- Be careful when using needles.
- Avoid skin contact with N2(l), it can cause cold burns.
- All materials that came into contact with transformed E. colishould be autoclaved before being disposed of.
Day 1
Inoculate 5 mL LB w/o salt in 50ml tube + 2 µL E.coli + 37 °C + 12:00:00 Overnight
Original:
Inoculate 5 ml LB w/o saltin a 50 ml culturing tube withE. coliand incubate overnight at 37°C / 250 rpm
Day 2
400 mL LB/ w/o salt in 1 Liter erlenmeyer flask + overnight culture
0.2 Genome copies per ml OD600
Original:
Next day, first thing in the morning, inoculate 400 ml LB w/o salt in a 1 l Erlenmeyer flask with enough of the overnight culture to reach OD600 of 0.2 and incubate for ~3 hours to OD600 0.5-1.0.
03:00:00
37 °C
0.5 Genome copies per ml OD600 <between> 1.0 Genome copies per ml OD600
0 °C ice 00:15:00
Original:
Transfer culture volume to 8 Greiner tubes of 50 ml and cool on ice for 15 minutes.
Centrifuge for 00:10:00 2000 g 4 °C
Discard supernatant and add5 mL of 10 % volume glycerol , resuspend the pellet in it and then add 45 mL of 10 % volume glycerol , shake by hand a few times to mix.
Centrifuge for 10 minutes at 2000g and 4°C.
Discard supernatant and add 5ml of 10% glycerol, resuspend the pellet in it and then add 45ml of 10% glycerol, shake by hand a few times to mix.
Put on ice for 10 minutes.
Centrifuge for 10 minutes at 2000g and 4°C.
Discard supernatant and resuspend the pellet in 1 ml ice-cold 10% glycerol, pool the contents of all 8 tubes into one tube.
Centrifuge for 5 minutes at 1500g and 4°C.
Discard supernatant and add 400 μl 10% glycerol (this will result in ~800 μl suspension).
Make aliquots of 40μl in pre-cooled eppendorf tubes and flash freeze with N2(l). Store at -80°C.