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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 2cm 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 2cm, 4cm, 6cm, 8cm 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 0.811g of anhydrous FeCl3 in 10ml H2O to obtain 0.5M solution
2. Add 1ml of FeCl3 0.5M solution to 50ml of 35% HClO4
3. store at room temperature in absence of sunlight
To perform the assay:
1. Measure the OD of your auxin producing cells and control cells at 600 nm to account for any difference in growth.
2. Spin down each cell sample and take an aliquot of supernatant to filter with ...
3. Add salkowski reagent to the filtered supernatant in a ratio of 2:1
4. Leave in the dark for 25-30 mins and then measure OD at 530 nm. (You should be able to see a visible colour change to pink/red if auxin is present.
