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Team:TU Delft/protocols/transformation
From 2010.igem.org
(New page: ==Transformation== ''Materials:'' - competent cells - LB medium (warmed to room temperature) - DNA ligation mix - LB plates containing 15-100 μg/mL antibiotic of choice, pre-warmed t...) |
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''Materials:'' | ''Materials:'' | ||
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- LB medium (warmed to room temperature) | - LB medium (warmed to room temperature) | ||
| - | - DNA ligation mix | + | - 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 37 °C |
| - | - | + | - shaking incubator at 37 °C. |
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1. Add 50-100 ng DNA into a 30 μL competent ''E.coli'', and mix gently. Do not mix by pipetting up and down! | 1. Add 50-100 ng DNA into a 30 μL competent ''E.coli'', and mix gently. Do not mix by pipetting up and down! | ||
| - | 2. Incubate tube vial on ice for | + | 2. Incubate tube vial on ice for 15 minutes. |
3. Heat-shocks the cells for exactly 5 minutes at 37 °C without shaking. | 3. Heat-shocks the cells for exactly 5 minutes at 37 °C without shaking. | ||
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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 800 μL of room temperature LB 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 | + | 7. Spin tube (2,000 rpm, 4 minutes), discard supernatant to leave no more than 100 μL, mix and plate on an agar plate. |
| - | more than 100 μL, | + | |
8. Incubate plates overnight at 37 °C. | 8. Incubate plates overnight at 37 °C. | ||
Latest revision as of 09:22, 12 September 2010
Transformation
Materials:
- competent cells
- LB 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 37 °C
- shaking incubator at 37 °C.
Protocol:
1. Add 50-100 ng DNA into a 30 μL competent E.coli, and mix gently. Do not mix by pipetting up and down!
2. Incubate tube vial on ice for 15 minutes.
3. Heat-shocks the cells for exactly 5 minutes at 37 °C without shaking.
4. Immediately transfer the tubes back to ice for 2 minutes.
5. Add 800 μL of room temperature LB medium.
6. Cap tube tightly and shake tube horizontally (225 rpm) at 37 °C for 1 hour.
7. Spin tube (2,000 rpm, 4 minutes), discard supernatant to leave no more than 100 μL, mix and plate on an agar plate.
8. Incubate plates overnight at 37 °C.
