Team:Imperial College London/Protocols Chemotaxis

From 2011.igem.org

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<h1>Phyto-Route</h1>
<h1>Phyto-Route</h1>
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<h2>Tryptone Broth</h2>
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<h2>Tryptone broth</h2>
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<hr style="color:#225323;"/>
<h2>PBS</h2>
<h2>PBS</h2>
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<hr style="color:#225323;"/>
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<h2>Motility Medium</h2>
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<h2>Motility medium</h2>
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<hr style="color:#225323;"/>
<h2>Preparation</h2>
<h2>Preparation</h2>
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<hr style="color:#225323;"/>
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<h2>Agar Plugin</h2>
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<h2>Agar plugin</h2>
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<hr style="color:#225323;"/>
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<h2>Semi-solid Agar</h2>
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<h2>Semi-solid agar</h2>
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<hr style="color:#225323;"/>
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<h2>Capillary Assay</h2>
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<h2>Capillary assay</h2>
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<hr style="color:#225323;"/>
<h2>Plant uptake</h2>
<h2>Plant uptake</h2>
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<p>-grow GFP+ E coli to exponential phase<br>
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<p>- Grow GFP-expressing <i>E. coli</i> to exponential phase<br>
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-spin down bacteria (5000rpm for 10min) and take off LB media<br>
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- Spin down bacteria (5000 rpm for 10 min) and remove LB medium supernatant.<br>
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-wash twice with wash buffer (5mM MES)<br>
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- Wash twice with wash buffer (5 mM MES).<br>
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-resuspend in wash buffer so that the bacteria are at OD 30<br>
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- Re-suspend in wash buffer so that the bacteria are at OD 30. <br>
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-put 10 Arabidopsis seedlings into 100ml of growth media each<br>
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- Put 10 Arabidopsis seedlings into 100 ml of growth media each. <br>
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-add bacteria to plant growth media, add the same amount of wash buffer to the negative control<br>
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- Add bacteria to plant growth media, add the same amount of wash buffer to the negative control. <br>
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-image after 12h and 24h</p>
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- Image after 12 h and 24 h.</p>
<p><b>Some notes</b><br>
<p><b>Some notes</b><br>
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- Some departments have very strong restrictions on bringing bacteria into dedicated plant growing areas<br>
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- Some departments have very strong restrictions on bringing bacteria into dedicated plant growing areas. <br>
- After talking to our health and safety officers, we decided to do all preparation and disposal of bacterial cultures as well as addition of the bacteria to plant media in biosafety hoods in dedicated level 1 or 2 laboratories so that there was no danger of dispersing the bacteria around the plant rooms.</p>
- After talking to our health and safety officers, we decided to do all preparation and disposal of bacterial cultures as well as addition of the bacteria to plant media in biosafety hoods in dedicated level 1 or 2 laboratories so that there was no danger of dispersing the bacteria around the plant rooms.</p>
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Revision as of 16:18, 18 September 2011




Protocols

This page lists all the protocols used in our project. We have classified them into five main categories as follow.




Phyto-Route

Tryptone broth


PBS


Motility medium


Preparation


Agar plugin


Semi-solid agar


Capillary assay


Plant uptake


- Grow GFP-expressing E. coli to exponential phase
- Spin down bacteria (5000 rpm for 10 min) and remove LB medium supernatant.
- Wash twice with wash buffer (5 mM MES).
- Re-suspend in wash buffer so that the bacteria are at OD 30.
- Put 10 Arabidopsis seedlings into 100 ml of growth media each.
- Add bacteria to plant growth media, add the same amount of wash buffer to the negative control.
- Image after 12 h and 24 h.

Some notes
- Some departments have very strong restrictions on bringing bacteria into dedicated plant growing areas.
- After talking to our health and safety officers, we decided to do all preparation and disposal of bacterial cultures as well as addition of the bacteria to plant media in biosafety hoods in dedicated level 1 or 2 laboratories so that there was no danger of dispersing the bacteria around the plant rooms.