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Team:Amsterdam/Notebook/Protocols/Making electrocompetent Cells
From 2011.igem.org
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(→Preparation of electrocompetent cells) |
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*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. | + | *10% glycerol. Sterilise by passing through 0.22-µm filter. Store at 4%deg;C |
===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 | ||
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*Centrifuge at 4000g at 4°C for 10 minutes in a flat bottom centrifuge bottle | *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. | *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 | + | *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 | *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 | + | *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 | ** This step can be repeated up to 3 times. Repeated cycles can increase competence, but will decrease yield | ||
| - | *After | + | *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 | ||
| + | * Hold on ice for 30 minutes | ||
| + | * Heat shock 60 sec at 42°C | ||
| + | * Add 200 μl [[Team:Amsterdam/Notebook/Protocols#Making SOB Medium (P3.)|SOC]] | ||
| + | * shake at 37°C for 2 hours in 14 ml aerated tubes | ||
| + | * Plate 20 μl and 200 on AMP plates using a sterile plate spreader | ||
| + | ** Transformation efficiency is number of colonies / (ng of DNA x partition of total volume plated) x 1000 | ||
| + | ** Competence should be at least 5x10<sup>7</sup>. The higher the better. | ||
{{:Team:Amsterdam/Footer}} | {{:Team:Amsterdam/Footer}} | ||
Revision as of 09:37, 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%deg;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
- Hold on ice for 30 minutes
- Heat shock 60 sec at 42°C
- Add 200 μl SOC
- shake at 37°C for 2 hours in 14 ml aerated tubes
- Plate 20 μl and 200 on AMP plates using a sterile plate spreader
- Transformation efficiency is number of colonies / (ng of DNA x partition of total volume plated) x 1000
- Competence should be at least 5x107. The higher the better.
