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Team:Calgary/Sandbox/Notebook/Protocols/BacterialTransformation
From 2013.igem.org
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<h2>Protocol</h2> | <h2>Protocol</h2> | ||
<ol> | <ol> | ||
| - | + | <li> Thaw 100 μL of competent cells (per transformation) on ice just before they are needed;</li> | |
| - | + | <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> Place on ice for 5 minutes;</li> | |
| - | + | <li> Add 250ul SOC medium to each tube;</li> | |
| - | + | <li> Incubate for 30 to 60 minutes with shaking at 37 degrees Celsius (note that for Kanamycin containing plasmids always use one hour);</li> | |
| - | + | <li> Spin down to remove all supernatant except approximately 100 μL;</li> | |
| - | + | <li> Plate approximately 50 μL on antibiotic plates;</li> | |
| - | + | <li> Grow overnight at 37 degrees Celsius.</li> | |
| - | + | </ol> | |
</section> | </section> | ||
</html> | </html> | ||
Revision as of 04:45, 16 September 2013
Bacterial Transformation
Protocol
- 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 degrees Celsius (note that for Kanamycin containing plasmids always use one hour);
- Spin down to remove all supernatant except approximately 100 μL;
- Plate approximately 50 μL on antibiotic plates;
- Grow overnight at 37 degrees Celsius.
