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Team:Amsterdam/Notebook/Protocols/Making electrocompetent Cells
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===Materials=== | ===Materials=== | ||
| + | *Detergent-free, sterile glassware and plasticware. We use dedicated centrifuge tubes that are only used for preparation of competent cells | ||
| + | *Table-top OD600nm spectrophotometer | ||
| + | *[[Team:Amsterdam/Notebook/Protocols#Making SOB Medium (P2.)|SOB]] ''WITHOUT'' MgCl | ||
| + | |||
===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 not that ''E. coli'' growth rate is significantly reduced due to the absence of Magnesium | **please not that ''E. coli'' growth rate is significantly reduced due to the absence of Magnesium | ||
Revision as of 08:42, 21 September 2011
Contents |
Preparation of electrocompetent cells
Overview
We use a protocol commonly used in our host lab provided by Diewertje Piebes.
Materials
- 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
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 not that E. coli growth rate is significantly reduced due to the absence of Magnesium
