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Team:Imperial College London/Protocols Auxin
From 2011.igem.org
Protocols
This page lists all the protocols used in our project. We have classified them into five main categories as follow.
Auxin Xpress
Effect of auxin on plants
Observation of root length in phytogels.
- Prepare half-MS phytogels (see plant protocols).
- Mark spots 2 cm apart from each other where you are going to plant the seeds.
- Inject auxin dissolved in 70% ethanol at one of these points. The phytogel is very soft so you can inject the solution directly into the gel using a Gilson pipette. Use concentrations of 0.0001, 0.001 and 0.01 mM of IAA.
- Seed DR5 reporter line seeds at distances of 2 cm, 4 cm, 6 cm, 8 cm from the auxin.
Split-root experiment
- Prepare horizontally-split plates. Pour regular half-MS phytogel into one half and phytogel containing 0.0001, 0.001 and 0.01mM phytogel into the other half. Pour only regular phytogel into the control plates.
- Take a DR5 reporter line seedling, previously grown in liquid culture and plant with one half of the roots in one half of the plate and the rest of the roots in the other half.
Salkowski
To make reagent:
1. Dissolve 0.811 g of anhydrous FeCl3 in 10 ml H2O to obtain 0.5 M solution
2. Add 1 ml of FeCl3 0.5 M solution to 50 ml of 35% HClO4
3. Store at room temperature in absence of sunlight
To perform the assay:
1. Measure the O.D. of your auxin-producing cells and control cells at 600 nm wavelenght to account for any difference in growth.
2. Spin down each cell sample and take an aliquot of supernatant to filter with 0.2 µm pore-size filters.
3. Add Salkowski reagent to the filtered supernatant in a ratio of 2:1
4. Leave in the dark for 25-30 min and then measure O.D. at 530 nm. (You should be able to see a visible colour change to pink/red if auxin is present.
