Team:Imperial College London/Protocols Auxin

From 2011.igem.org

(Difference between revisions)
Line 7: Line 7:
<h1>Auxin Xpress</h1>
<h1>Auxin Xpress</h1>
-
<h2>Effect of Auxin on Plants</h2>
+
<h2>Effect of auxin on plants</h2>
<hr style="color:#225323;"/>
<hr style="color:#225323;"/>
-
<p><b>Observation of root length in phytogels</b><br>
+
<p><b>Observation of root length in phytogels. </b><br>
-
-prepare half-MS phytogels (see plant protocols)<br>
+
- Prepare half-MS phytogels (see plant protocols).<br>
-
-mark spots 2cm apart from each other where you are going to plant the seeds<br>
+
- Mark spots 2 cm 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>
+
- 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.</p>
+
- Seed DR5 reporter line seeds at distances of 2 cm, 4 cm, 6 cm, 8 cm from the auxin.</p>
-
<h2>Split Root Experiment</h2>
+
<h2>Split-root experiment</h2>
<hr style="color:#225323;"/>
<hr style="color:#225323;"/>
-
<p>-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>
+
<p>- 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.</p>
+
- 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.</p>
<h2>Salkowski</h2>
<h2>Salkowski</h2>
-
<p><b>To make reagent:<b></p>
+
<p><b>To make reagent:</b></p>
-
<p>1. Dissolve 0.811g of anhydrous FeCl3 in 10ml H2O to obtain 0.5M solution</p>
+
<p>1. Dissolve 0.811 g of anhydrous FeCl3 in 10 ml H2O to obtain 0.5 M solution</p>
-
<p>2. Add 1ml of FeCl3 0.5M solution to 50ml of 35% HClO4 </p>
+
<p>2. Add 1 ml of FeCl3 0.5 M solution to 50 ml of 35% HClO4 </p>
-
<p>3. store at room temperature in absence of sunlight</p>
+
<p>3. Store at room temperature in absence of sunlight</p>
-
<p><b>To perform the assay:<b></p>
+
<p><b>To perform the assay:</b></p>
-
<p>1. Measure the OD of your auxin producing cells and control cells at 600 nm to account for any difference in growth.</p>
+
<p>1. Measure the O.D. of your auxin-producing cells and control cells at 600 nm wavelenght to account for any difference in growth.</p>
-
<p>2. Spin down each cell sample and take an aliquot of supernatant to filter with ...</p>
+
<p>2. Spin down each cell sample and take an aliquot of supernatant to filter with 0.2 </p>
-
<p>3. Add salkowski reagent to the filtered supernatant in a ratio of 2:1</p>
+
<p>3. Add Salkowski reagent to the filtered supernatant in a ratio of 2:1</p>
-
<p>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. </p>  
+
<p>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. </p>  
<hr style="color:#225323;"/>
<hr style="color:#225323;"/>
-
<h2>HPLC</h2>
+
<h2>HPLC (High-performance liquid chromatography)</h2>
<hr style="color:#225323;"/>
<hr style="color:#225323;"/>
-
<h2>Soil Erosion Experiment</h2>
+
<h2>Soil erosion experiment</h2>
<hr style="color:#225323;"/>
<hr style="color:#225323;"/>
</body>
</body>
</html>
</html>

Revision as of 16:26, 18 September 2011



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

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

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)

Soil erosion experiment

Retrieved from "http://2011.igem.org/Team:Imperial_College_London/Protocols_Auxin"