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Team:TU Delft/protocols/transformation2
From 2010.igem.org
(New page: ==Transformation== ''Materials:'' - competent cells - SOC medium (warmed to room temperature) - DNA (e.g. plasmid, ligation mix) - LB plates containing 15-100 μg/mL antibiotic of cho...) |
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| - | + | =Transformation= | |
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''Materials:'' | ''Materials:'' | ||
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- SOC medium (warmed to room temperature) | - SOC medium (warmed to room temperature) | ||
| - | - DNA | + | - Plasmid DNA or DNA ligation mix |
| - | - LB plates containing 15-100 μg/mL antibiotic of choice, pre-warmed to 37 °C | + | - LB agar plates containing 15-100 μg/mL antibiotic of choice, pre-warmed to 37 °C |
| - | - | + | - water bath at 42 °C |
| - | - | + | - shaking incubator at 37 °C. |
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4. Immediately transfer the tubes back to ice for 2 minutes. | 4. Immediately transfer the tubes back to ice for 2 minutes. | ||
| - | 5. Add | + | 5. Add 250 μL of room temperature SOC medium. |
6. Cap tube tightly and shake tube horizontally (225 rpm) at 37 °C for 1 hour. | 6. Cap tube tightly and shake tube horizontally (225 rpm) at 37 °C for 1 hour. | ||
| - | 7. Plate from each tube 100 μL on an agar plate containing antibiotic | + | 7. Plate from each tube 100 μL on an agar plate containing antibiotic. |
8. Incubate plates overnight at 37 °C. | 8. Incubate plates overnight at 37 °C. | ||
Latest revision as of 09:26, 12 September 2010
Transformation
Materials:
- competent cells
- SOC medium (warmed to room temperature)
- Plasmid DNA or DNA ligation mix
- LB agar plates containing 15-100 μg/mL antibiotic of choice, pre-warmed to 37 °C
- water bath at 42 °C
- shaking incubator at 37 °C.
Protocol:
1. Add 50-100 ng DNA into a 15 μL competent E.coli, and mix gently. Do not mix by pipetting up and down!
2. Incubate tube vial on ice for 30 minutes.
3. Heat-shocks the cells for exactly 30 seconds at 42 °C without shaking.
4. Immediately transfer the tubes back to ice for 2 minutes.
5. Add 250 μL of room temperature SOC medium.
6. Cap tube tightly and shake tube horizontally (225 rpm) at 37 °C for 1 hour.
7. Plate from each tube 100 μL on an agar plate containing antibiotic.
8. Incubate plates overnight at 37 °C.
