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Team:Amsterdam/Notebook/Protocols/Making electrocompetent Cells
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=Preparation of electrocompetent cells= | =Preparation of electrocompetent cells= | ||
| - | ==Overview== | + | ===Overview=== |
| - | 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 | + | 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 is a 1-2 orders of magnitude higher level of competence. 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=== | ||
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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. Sterilise by passing through 0.22-µm filter. Store at 4 | + | *10% glycerol. Sterilise by passing through 0.22-µm filter. Store at 4°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 | ||
| Line 22: | Line 23: | ||
*Once again decant supernatant and resuspend pellet in 50-100 ml of ice-cold 10% glycerol | *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 one final centrifugation step resuspend cells in | + | *After one final centrifugation step resuspend cells in 0.5ml 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 | *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 | *Store at -80°C indefinitely | ||
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* Transform 50 μl of cells with 10 pg of standard pUC19 | * Transform 50 μl of cells with 10 pg of standard pUC19 | ||
** This is 1 μl of standard pUC19 Invitrogen plasmid | ** This is 1 μl of standard pUC19 Invitrogen plasmid | ||
| - | * | + | *Incubate on ice for 30 minutes |
| - | * | + | *Electroshock at 1250 volt. Monitor time |
| - | * | + | **electroporation time should be higher than ~4.5. |
| - | * shake at 37°C | + | *add 900 µl of prewarmed SOC, pipette up and down and transfer to roundbottom aerated tube |
| - | * | + | *shake for 90 minutes at 37°C |
| + | *transfer to 1.5 ml eppendorf tube | ||
| + | *spin down at 5Krpm for 5 minutes | ||
| + | *decant supernatant | ||
| + | *resuspend pellet in last drop of SOC and plate on selective LB-agar plates using a sterile plate spreader | ||
** Transformation efficiency is number of colonies / (ng of DNA x partition of total volume plated) x 1000 | ** 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. | ** Competence should be at least 5x10<sup>7</sup>. The higher the better. | ||
Latest revision as of 12:22, 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 is a 1-2 orders of magnitude higher level of competence. 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 0.5ml 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
- Incubate on ice for 30 minutes
- Electroshock at 1250 volt. Monitor time
- electroporation time should be higher than ~4.5.
- add 900 µl of prewarmed SOC, pipette up and down and transfer to roundbottom aerated tube
- shake for 90 minutes at 37°C
- transfer to 1.5 ml eppendorf tube
- spin down at 5Krpm for 5 minutes
- decant supernatant
- resuspend pellet in last drop of SOC and plate on selective LB-agar 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.
