Team:Calgary/Notebook/Protocols/Process13
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<ol> | <ol> | ||
<li> Thaw 100 μL of competent cells (per transformation) on ice just before they are needed</li> | <li> Thaw 100 μL of competent cells (per transformation) on ice just before they are needed</li> | ||
- | <li> Add DNA (max | + | <li> Add DNA (max 20μl) thawed cells and mix by flicking the side of the tube. Leave on ice for 30 minutes</li> |
<li> Heat shock 5 minutes at 37 degrees Celsius</li> | <li> Heat shock 5 minutes at 37 degrees Celsius</li> | ||
<li> Place on ice for 5 minutes </li> | <li> Place on ice for 5 minutes </li> | ||
<li> Add 250ul SOC medium to each tube</li> | <li> Add 250ul SOC medium to each tube</li> | ||
- | <li> Incubate for 30 to 60 minutes with shaking at 37 | + | <li> Incubate for 30 to 60 minutes with shaking at 37°C. (Note that for Kanamycin containing plasmids always use one hour)</li> |
<li> Spin down to remove all supernatant except approximately 100 μL</li> | <li> Spin down to remove all supernatant except approximately 100 μL</li> | ||
<li> Plate approximately 30 μL on each of two antibiotic plates </li> | <li> Plate approximately 30 μL on each of two antibiotic plates </li> | ||
- | <li> Grow overnight at 37 | + | <li> Grow overnight at 37°C </li> |
</ol> | </ol> | ||
<p> For this protocol we used a couple of controls | <p> For this protocol we used a couple of controls |
Latest revision as of 04:21, 29 September 2011
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Bacterial Transformation
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- Thaw 100 μL of competent cells (per transformation) on ice just before they are needed
- Add DNA (max 20μl) thawed cells and mix by flicking the side of the tube. Leave on ice for 30 minutes
- Heat shock 5 minutes at 37 degrees Celsius
- Place on ice for 5 minutes
- Add 250ul SOC medium to each tube
- Incubate for 30 to 60 minutes with shaking at 37°C. (Note that for Kanamycin containing plasmids always use one hour)
- Spin down to remove all supernatant except approximately 100 μL
- Plate approximately 30 μL on each of two antibiotic plates
- Grow overnight at 37°C
For this protocol we used a couple of controls
- Positive Control - pBluescript in TOP10 cells on ampicillin plates
- Negative Control - TOP10 cells grown on ampcillin plates
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