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Team:Amsterdam/Notebook/Protocols/Making electrocompetent Cells
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We use a protocol commonly used in our host lab provided by [https://2011.igem.org/Team:Amsterdam/Team/Advisors Diewertje Piebes]. The main advantage of electrocompetent cells compared to chemically competent cells are a higher level of competence (1-2 log higher). The disadvantage, though, is the high price of electroporation cuvettes and the implications this has on maximum number of transformations that can be performed in a single experiment. | We use a protocol commonly used in our host lab provided by [https://2011.igem.org/Team:Amsterdam/Team/Advisors Diewertje Piebes]. The main advantage of electrocompetent cells compared to chemically competent cells are a higher level of competence (1-2 log higher). The disadvantage, though, is the high price of electroporation cuvettes and the implications this has on maximum number of transformations that can be performed in a single experiment. | ||
Revision as of 09:41, 21 September 2011
Contents |
Preparation of electrocompetent cells
Overview
We use a protocol commonly used in our host lab provided by Diewertje Piebes. The main advantage of electrocompetent cells compared to chemically competent cells are a higher level of competence (1-2 log higher). The disadvantage, though, is the high price of electroporation cuvettes and the implications this has on maximum number of transformations that can be performed in a single experiment.
Materials
- Prechilled detergent-free, sterile glassware and plasticware. We use dedicated centrifuge tubes that are only used for preparation of competent cells
- Table-top OD600nm spectrophotometer
- SOB WITHOUT MgCl
- 10% glycerol. Sterilise by passing through 0.22-µm filter. Store at 4°C
Procedure
- Prepare TOP10 preculture by inoculating 3ml of LB medium with a -80°C TOP10 glycerol stock into and shake overnight at 37°C
- Inoculate 200 ml of SOB containing no magnesium with 3 ml of preculture and grow in a 37°C shaker to an OD600 of 0.6-0.8
- please note that E. coli growth rate is significantly reduced due to the absence of Magnesium
- it is possible to add more preculture if you are in a hurry, however, it is not recommended
- Prewarmed medium can shorten preparation time by up to one hour
- Centrifuge at 4000g at 4°C for 10 minutes in a flat bottom centrifuge bottle
- Note that from this point on the cells, media, bottles and glassware will have to be kept on ice at all times!! If possible it is recommended to work in a cold room.
- decant supernatant and resuspend pellet in 50-100 ml of ice-cold 10% glycerol
- Centrifuge at 4000g at 4°C for 10 minutes in a flat bottom centrifuge bottle
- Once again decant supernatant and resuspend pellet in 50-100 ml of ice-cold 10% glycerol
- This step can be repeated up to 3 times. Repeated cycles can increase competence, but will decrease yield
- After one final centrifugation step resuspend cells in (2ml???) of ice-cold 10% glycerol
- Aliquot 50 μl samples to sterile, prechilled, 1.5 ml or 2 ml eppendorf tubes and flash freeze by immersion in liquid nitrogen
- Store at -80°C indefinitely
- Test competence (see below)
Measurement of competence
- Transform 50 μl of cells with 10 pg of standard pUC19
- This is 1 μl of standard pUC19 Invitrogen plasmid
