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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 assay
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.
HPLC (High-performance liquid chromatography)
To extract IAA from cell filtrate:
1. Centrifuge 10ml of culture at 5000rpm for 10 minutes.
2. Filter supernatant with 0.2 μm pore filter.
3. Acidify the filtrate through the drop-wise addition of 4M HCl in order to make the pH of the solution 4.
4. Add three volumes of ethyl acetate and shake vigorously.
5. Let the mixture stand until a phase between the two liquids is visible.
6. Remove the ethyl acetate layer and discard the rest.
7. Evaporate extracts to dryness at 39°C under vacuum.
8. Dissolve the precipitate in 2 ml of methanol.
9. HPLC analysis was performed with 10 μl aliquots.
To make solvent for HPLC:
Mix H2O with acetonitrile and acetic acid in a ratio of 85:15:1 (H2O: acetonitrile: acetic acid)
To analyze with HPLC:
Analyze under isocratic conditions at a flow rate of 1ml/min.
Extraction for LCMS (Liquid chromatography mass spectrometry)
1. Centrifuge 10ml of culture at 5000rpm for 10 minutes.
2. Filter supernatant with 0.2 μm pore filter.
3. Acidify the filtrate through the drop-wise addition of 4M HCl in order to make the pH of the solution 4.
4. Add two volumes of ethyl acetate and shake vigorously.
5. Let the mixture stand until a phase between the two liquids is visible.
6. Remove the ethyl acetate layer and discard the rest.
7. Add 3/5 volumes of K2HPO4 and shake vigorously.
8. Pipette off the ethyl acetate layer and discard the rest.
9. Add Mg2SO4 to absorb water until it not longer dissolves in the extract
10. Centrifuge the samples at 5000 rpm for 10 minutes.
11. Carefully decant the ethyl acetate extract without Mg2SO4 precipitate.
12. Evaporate extracts to dryness at 39°C under vacuum.
13. Dissolve the precipitate in 1 ml of 15% acetonitrile.
14. Filter extracts through a 0.45 μm pore filter.
