Team:Imperial College London/Project/Arabidopsis/Protocols
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<h1>Auxin uptake protocol</h1> | <h1>Auxin uptake protocol</h1> | ||
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. <br> | 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. <br> | ||
- | - To test auxin sensitivity, | + | - To test auxin sensitivity, Arabidopsis seeds were sown onto medium as given above and supplemented with 0, 0.00001, 0.0001, 0.01, 1, 100, 10000uM indole-3-acetic acid (IAA). <br> |
- Medium preparation and seed sowing occurred under 0.5 pE m-2 sec-l incandescent light to minimize | - Medium preparation and seed sowing occurred under 0.5 pE m-2 sec-l incandescent light to minimize | ||
- | photooxidation of IAA. | + | photooxidation of IAA. <br> |
- | - Growing is done at | + | - Growing is done at 23°C in darkness for three days<br> |
- | - 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. <br><br> | + | - 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. <br> |
+ | -Plants were transferred to light for a further six days<br><br> | ||
Some notes<br><br> | Some notes<br><br> | ||
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-put 10 Arabidopsis seedlings into 100ml of growth media each<br> | -put 10 Arabidopsis seedlings into 100ml of growth media each<br> | ||
-add bacteria to plant growth media, add the same amount of wash buffer to the negative control<br> | -add bacteria to plant growth media, add the same amount of wash buffer to the negative control<br> | ||
- | -image after 12h and 24h<br> | + | -image after 12h and 24h<br><br> |
+ | Some notes<br><br> | ||
+ | - 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. <br> | ||
+ | - 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 <br> | ||
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Revision as of 09:30, 10 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 minutes per tube (mix well)
- Remove 70% EtOH and replace with 500µl 50% bleach
- 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
- add 10g sucrose (normally from 1% solution)
- 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 at constant light conditions
- Grow seedlings for 5-6 days
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, Arabidopsis seeds were sown onto medium as given above and supplemented with 0, 0.00001, 0.0001, 0.01, 1, 100, 10000uM 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.
- Growing is done at 23°C in darkness for three days
- 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.
-Plants were transferred to light for a further six days
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.
Glycerol stock protocol
- obtain the bacterial pellet from centrifugation- resuspend the pellet with _microl dH20
- add _microl of 80% glycerol in each eppendorf.
- mix bacteria in 80% glycerol by resuspending the liquid many times
Plant uptake of E coli
-grow GFP+ E coli to exponential phase-spin down bacteria (5000rpm for 10min) and take off LB media
-wash twice with wash buffer (5mM MES)
-resuspend in wash buffer so that the bacteria are at OD 30
-put 10 Arabidopsis seedlings into 100ml of growth media each
-add bacteria to plant growth media, add the same amount of wash buffer to the negative control
-image after 12h and 24h
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