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Team:Harvard/vectors/agrotoplants
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| + | <h2>Protocol: Flower-Dip Transformation of Arabidopsis</h2> | ||
| + | <p>When plants have been growing for 4-6 weeks and have flowers it's time to do a flower dip!</p> | ||
| + | <img src="https://static.igem.org/mediawiki/2010/e/e9/Arabisopsisreadytodip.JPG" width="600px" /> | ||
| + | <p>We followed the procedure outlined in the open access paper <a href="http://www.plantmethods.com/content/2/1/16">Logemann et. al.</a> "An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transfromation protocol."</P | ||
| + | <p>First, YEB lawn plates of the Agrobacteria transformed with each of our transformation vectors were scraped and the cells were resuspended in 50mL conical tubes containing 30 mL of YEB or LB.</p> | ||
| + | <img src="https://static.igem.org/mediawiki/2010/1/12/Scraping_agro.png" width="600px" /> | ||
| + | <p>This suspension of Agrobacteria is then mixed with 120mL of the surfactant sucrose solution. Per 120 mL dip solution:</p> | ||
| + | <ul><li>6 grams sucrose</li> | ||
| + | <li>36 uL of L-77 surfactant</li> | ||
| + | <li>120 mL of sterile water</li> | ||
| + | </ul> | ||
| + | |||
| + | <p>The flower pot is then simply turned upside down and the flowers swished in the surfactant/sucrose/agrobacterium solution for ten seconds</p> | ||
| + | |||
| + | <img src="https://static.igem.org/mediawiki/2010/7/77/Flowerdip.png" width="600px" /> | ||
| + | |||
| + | <p>The dipped plants are then kept in a humidified chamber for 2-4 days and dipped for a second time in freshly made bacterial solution after 4-7 days. After the second dip plants are monitored for several weeks as the seed pods form and dry, at which point the seeds are harvested, dried, and planted on selective agar plates for analysis of transformation efficiency.</p> | ||
</div> | </div> | ||
</div> | </div> | ||
Revision as of 17:44, 12 October 2010
abstract lab notebook agrobacteria and plant engineering meet the vectors plant transformation parts & primers references
Protocol: Flower-Dip Transformation of Arabidopsis
When plants have been growing for 4-6 weeks and have flowers it's time to do a flower dip!
We followed the procedure outlined in the open access paper Logemann et. al. "An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transfromation protocol."
First, YEB lawn plates of the Agrobacteria transformed with each of our transformation vectors were scraped and the cells were resuspended in 50mL conical tubes containing 30 mL of YEB or LB.
This suspension of Agrobacteria is then mixed with 120mL of the surfactant sucrose solution. Per 120 mL dip solution:
- 6 grams sucrose
- 36 uL of L-77 surfactant
- 120 mL of sterile water
The flower pot is then simply turned upside down and the flowers swished in the surfactant/sucrose/agrobacterium solution for ten seconds
The dipped plants are then kept in a humidified chamber for 2-4 days and dipped for a second time in freshly made bacterial solution after 4-7 days. After the second dip plants are monitored for several weeks as the seed pods form and dry, at which point the seeds are harvested, dried, and planted on selective agar plates for analysis of transformation efficiency.
