Experiment Metadata
Experiment Name:
FatB Induction Experiment
Goal:
This experiment tests the consequence of a mutation at the FatB gene (At1g08510) in the wound-response of Arabidopsis. The FatB mutant allele (fatb-KD) was obtained from Dr. Katayonn Dehesh, University of California, Davis, Davis, CA. The fatb-KD allele is in the Ws background, and is described in Plant Cell 2003, vol 15, 1020-1033.
Growth Conditions:
The standardized growth conditions are as follows:
1. Seeds (between 14 and 16) are sown on media in 100 x 100 x 15mm square Falcon Petri Dishes (Fisher Scientific, catalogue #08-757-11A). 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 Micro-pore 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 12-days - this growth room condition is explained in Table I.
6. On the 13th day, at 11:30 am local time, plates were uncovered in the growth room and leaves were wounded by piercing with an 18 gauge sterile needle, each leaf has approximately 3 piercing. Plates were then recovered with their lids and wrapped with Micro-pore tape (3M Health Care, catalogue #1530-0). Uncovering, wounding and resealing the plates took 3 minutes for each plate. All plants, including the non-wounded plants, were exposed to the air for this amount of time. Plates were then placed vertically in Plexi-glass holders at 'normal' conditions.
7. On 13th day, at 1:30 pm (2 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 |
|---|---|
| 09/25/06 | Sowing of Seeds |
| 09/29/06 | Move into growth room |
| 10/11/06 | Induction |
| 10/11/06 | Harvest |
| 11/07/06 | Shipment to analytical labs |