"
Team:Calgary/Sandbox/Notebook/Protocols/BacterialTransformation
From 2013.igem.org
(Difference between revisions)
| Line 6: | Line 6: | ||
<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>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>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>Heat shock 5 minutes at 37 degrees Celsius</li> |
| - | <li>Place on ice for 5 minutes | + | <li>Place on ice for 5 minutes</li> |
| - | <li>Add 250ul SOC medium to each tube | + | <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>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>Spin down to remove all supernatant except approximately 100 μL</li> |
| - | <li>Plate approximately 50 μL on antibiotic plates | + | <li>Plate approximately 50 μL on antibiotic plates</li> |
| - | <li>Grow overnight at 37 degrees Celsius | + | <li>Grow overnight at 37 degrees Celsius</li> |
</ol> | </ol> | ||
</section> | </section> | ||
</html> | </html> | ||
Revision as of 04:53, 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
