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Team:Amsterdam/Notebook/Protocols/Making electrocompetent Cells
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==Overview== | ==Overview== | ||
| - | We use a protocol commonly used in our host lab provided by [https://2011.igem.org/Team:Amsterdam/Team/Advisors Diewertje Piebes]. | + | 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. |
===Materials=== | ===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 | *Table-top OD600nm spectrophotometer | ||
*[[Team:Amsterdam/Notebook/Protocols#Making SOB Medium (P2.)|SOB]] '''WITHOUT''' MgCl | *[[Team:Amsterdam/Notebook/Protocols#Making SOB Medium (P2.)|SOB]] '''WITHOUT''' MgCl | ||
| - | + | *10% glycerol. Filter sterilized | |
===Procedure=== | ===Procedure=== | ||
*Prepare TOP10 preculture by inoculating 3ml of LB medium with a -80°C TOP10 glycerol stock into and shake overnight at 37°C | *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 | *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 | + | **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 10% ice-cold 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 10% ice-cold glycerol | ||
| + | ** This step can be repeated up to 3 times. Repeated cycles can increase competence, but will decrease yield | ||
| + | *After a | ||
Revision as of 09:25, 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. Filter sterilized
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 10% ice-cold 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 10% ice-cold glycerol
- This step can be repeated up to 3 times. Repeated cycles can increase competence, but will decrease yield
- After a
