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Team:Imperial College London/Project/Arabidopsis/Protocols
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<h1>Seedling protocol</h1> | <h1>Seedling protocol</h1> | ||
Revision as of 10:18, 3 August 2011
Seedling protocol
- Weigh in appr. 50mg of arabidopsis seeds in eppendorf tube (one tube per 250 ml erlenmayer flask)- Wash with 500 µl 70% EtOH for appr. 4-5 (or 2x2) minutes per tube (mix well)
- Remove 70% EtOH and replace with 500µl 50% bleach (alternatively 5% Calcium hypochloride)
- Incubate for 20 minutes
- Wash several times with sterile ddH2O to remove bleach x3
- Vernalize seeds for 2-3 days
Prepare sterile medium
- Half strength Murashige salt (2.1g per liter ddH2O)
- Add 0.546g MES salt (buffer) per liter of media
- Adjust pH to 5.7-5.8 using 2M KOH
- If required add 10g sucrose (e.g. 1% solution) – yes
- Add 1% agarose = 10g/litre if making phytogel
- Distribute into erlenmayer flasks (125 ml/250ml flask)
- Autoclave for at least 15 minutes
Some notes
- Growth conditions : flasks on a shaker at appr. 200 rpm. Either under long day or constant light conditions
- Grow seedlings for 5-6 days
Bacteria uptake protocol
Overview : Bacteria is taken into the root plant will express GFP. At higher concentration of E.coli, GFP might be expressing more, however higher bacteria concentration might defect the plant cell.- 10 ml of GFP. coli or GFPyeast preparation at a cell density of 50 A600 units was then added into the 250 ml hydroponic culture.
- After an overnight incubation at room temperature, roots were washed with deionized water and analyzed by CLSM.
- We can vary the concentration of E.coli by putting 0 (control), 5, 10, 20, and 40 ml of E.coli respectively
- For analysis of Arabidopsis root section by CLSM, visually assessed roots regions showing high fluorescence are excised (5–10 mm long), washed and embedded in 3% agarose.
- Hand-cut cross sections are transferred into curved slides, washed thoroughly with deionized water and analyzed by CLSM
Some notes
- Any work involving E coli will take place in the teaching labs and the plant growth room will only be used to grow plants in individual, sealed flasks: E coli will be added to media in the teaching labs and media change will also take place in the teaching labs to ensure containment of the bacteria.
- To ensure that no E coli get into the water ways in the plant rooms, we will dispose of the bacteria in the teaching labs by filling them into flasks, applying vircon and autoclaving the solution
Auxin uptake protocol
Overview : synthetic auxin is used to see the effect of Arabidopsis's root growth. Variation in auxin concentrations is applied to see the sensitivity of auxin in arabidopsis.- To test auxin sensitivity, sibling families were sown onto medium as given above and supplemented with O, 0.0001, 0.001, 0.01, 0.1, 1.0, 10, or 100 pM indole-3-acetic acid (IAA).
- Medium preparation and seed sowing occurred under 0.5 pE m-2 sec-l incandescent light to minimize photooxidation of IAA. We use the siblings of Wt arabidopsis instead which is subjected to the same condition 1 day before the experiment.
- Growing is done at 23OC in darkness in two randomized complete blocks.
- After 3 days, hypocotyl and root lengths were measured on 10 plantslreplication. Data were normalized to lengths as a percentage of the control treatment and subjected to analysis of variance.
Some notes
- Concentrations of IAA causing 50% inhibition of root and hypocotyl growth (Isow) ere calculated for each replication by solving regression equations with y = y intercept + 2.
