Latest revision as of 16:39, 11 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

Auxin concentration gradient effect on plants

-prepare half-MS phytogels (see above)
-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 auxin uptake

-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.

@@ Line 24: / Line 24: @@
 - Growth conditions : flasks on a shaker at appr. 200 rpm at constant light conditions<br>
-- Grow seedlings for 5-6 days<br>
+- Grow seedlings for 5-6 days<br><br>
-</html>
-<html>
-<h1>Bacteria uptake protocol</h1>
-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.<br>
-- 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.<br>
-- After an overnight incubation at room temperature, roots were washed with deionized water and analyzed by CLSM.<br>
-- We can vary the concentration of E.coli by putting 0 (control), 5, 10, 20, and 40 ml of E.coli respectively <br>
-- 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. <br>
-- Hand-cut cross sections are transferred into curved slides, washed thoroughly with deionized water and analyzed by CLSM<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>
-</html>
-<html>
 <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>
-- 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). <br>
+- 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
-photooxidation of IAA. We use the siblings of Wt arabidopsis instead which is subjected to the same condition 1 day before the experiment. <br>
+photooxidation of IAA.  <br>
-- Growing is done at 23OC in darkness in two randomized complete blocks.<br>
+- 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>
 - 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.<br>
+regression equations with y = y intercept + 2.<br><br>
@@ Line 60: / Line 46: @@
 - resuspend the pellet with _microl dH20 <br>
 - add _microl of 80% glycerol in each eppendorf. <br>
-- mix bacteria in 80% glycerol by resuspending the liquid many times <br>
+- mix bacteria in 80% glycerol by resuspending the liquid many times <br><br>
@@ Line 70: / Line 56: @@
 -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>
--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><br>
+<h1>Auxin concentration gradient effect on plants</h1>
+-prepare half-MS phytogels (see above)<br>
+-mark spots 2cm apart from each other where you are going to plant the seeds<br>
+-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.<br>
+-seed DR5 reporter line seeds at distances of 2cm, 4cm, 6cm, 8cm from the auxin.<br><br>
+<h1>Split-root auxin uptake</h1>
+-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.<br>
+-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.
 </html>

Team:Imperial College London/Project/Arabidopsis/Protocols

From 2011.igem.org