Experiment Metadata
Experiment Name:
Elo1 Induction Experiment
Goal:
This experiment is set-up to test the effect of the ectopic expression of the Arabidopsis ELO1 gene (At3g06460) in leaf tissues. Ectopic expression was achieved with a transgene driven by an inducible promoter, whose expression was activated by spraying transgenic plants with the insecticide, Intrepid 2F. Details of the transgene is available from Charles Dietrich and Ed Cahoon, Danforth Center, St. Louis, MO. The effect of ectopic ELO1 expression was compared to non-transgenic isogenic wild-type plants.
Growth Conditions:
The standardized growth conditions are as follows:
1. Seeds (between 17 and 19) are sown on media in 100 x 100 x 15mm square Falcon Petri Dishes (Becton Dickinson Labware, catalogue #351112). Seeds were arranged on the plates in a single horizontal line at the 1-cm mark from the top of the plate.
2. Each plate contains between 20 and 25-ml of sterile MS media containing 0.1% (w/v) sucrose.
3. Prior to sowing, seeds were sterilized by treating for 1-minute at room temperature with a 300-µl solution of 50% (v/v) ethanol, this solution was removed and replaced with a 300-µl solution consisting of 1% (v/v) Tween 20 (Fischer BioReagents, catalogue #BP33750), and 50% (v/v) bleach solution (Clorox), and incubated at room temperature for 10-minutes. The seeds were then washed with three changes of 0.3-ml of sterile water.
4. Upon sowing with seeds, the plates were wrapped with Micropore tape (3M Health Care, catalogue #1530-0), and then stored horizontally for 4-days at 4 oC, with illumination of 1 µmol/m2.
5. On the 5th day, plates were moved to the growth room, and held in a vertical position in Plexi-glass holders for 14-days - this growth room condition is labeled in Table I as the "normal" growth condition.
6. On the 15th day, at 10am local time, plates were uncovered in a sterile hood and sprayed with ~1 mL of 1/10,000 dilution of Intrepid 2F spray (sufficient to cover roots and aerial tissue) (INTREPID 2F INSECTICIDE, product code 88799 is a product of Dow Agrosciences, 9330 Zionsville Rd, Indiannapolis, IN46268). The plates were covered with lids and laid horizontal for 10 minutes before being wrapped with Mil-pore tape (3M Health Care, catalogue #1530-0). Plates were then placed vertically in Plexi-glass holders at 'normal' conditions.
7. On 16th (24 hours after induction), 17th (48 hours after induction) and 18th day (72 hours after induction) Petri plates were opened and the aerial portions of these plants were harvested immediately upon plate opening.
8. Upon harvesting, plant material was quenched by immersion in liquid nitrogen and stored at -70 0C.
Timeline
| Event Date | Event |
|---|---|
| 07/14/06 | Sowing of seeds |
| 7/17/2006 | Move into growth room |
| 8/1/2006 | Induction |
| 08/02/06, 08/03/06 and 08/04/06 | Harvest |
| 09/12/06 | Shipment to analytical labs |