Team:Imperial College London/Extras/Protocols/Chemotaxis

From 2011.igem.org

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<html>
<html>
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<h1>Chemotaxis Lab Protocols</h1>
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<body>
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<h2>27th of July</h2>
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The strain of E. coli with the copy of super-folded GFP that was incubated the previous night has shown limited/no growth in the prepared LB broth. This could have been caused by:<br>
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<h1>Phyto-Route</h1>
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-No innoculation<br>
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<h2>Tryptone Broth</h2>
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-Kanamycin concentration in LB broth was too high. We had used 85μg/ml.<br><br>
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<hr style="color:#225323;"/>
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<b>New protocol:</b><br>
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<h2>PBS</h2>
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50μg/ml is the recommended concentration. We will use 35-40μg/ml of kanamycin to speed up growth.</html>
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<html><h2>28th of July</h2>
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<h2>Motility Medium</h2>
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Transformation of cells with 6, 7 and 8:<br><br>
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-Let competent cell strain 5α thaw for around 10 minutes on ice.<br>
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<h2>Preparation</h2>
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-Add 2-3μl of DNA.<br>
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-Leave on ice for 20-25 minutes.<br>
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<h2>Agar Plugin</h2>
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-Heat shock cells at 42°C for 45 seconds.<br>
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<hr style="color:#225323;"/>
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-Leave on ice for 10 minutes.<br>
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<h2>Semi-solid Agar</h2>
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-Add 500μl of LB broth.<br>
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-Incubate for 1 hour at 37°C.<br>
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<h2>Capillary Assay</h2>
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-Centrifuge for 1 minute.<br>
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-Remove 100μl off the top of the eppendorf tube. Pour out the rest making sure that the pellet remains in the eppendorf tube.<br>
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-Re-suspend the cells in the 100μl LB broth solution that was removed in the previous step.
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-Add 5μl on a chloramphenicol agar plate (concentration of 35μg/ml).<br>
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-Add the rest of the sample to a second chloramphenicol agar plate.<br></html>
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<html><br><br>
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To make tryptone broth (bacterial growth before chemotaxis assays)for total volume of 1L:<br>
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-10g tryptone<br>
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-1000ml of 1X PBS<br>
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-autoclave<br>
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-1.14g kanamycin<br>
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<br>
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To make 1X PBS (phosphate buffer saline):<br>
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-in 800ml of distilled H<sub>2</sub>O<br>
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-8g of NaCl<br>
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-0.2g of KCl<br>
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-1.44g of Na<sub>2</sub>HPO<sub>4</sub><br>
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-0.24g of KH<sub>2</sub>PO<sub>4</sub><br>
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-adjust pH to 7.4<br>
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-adjust volume 1L with additional distilled H<sub>2</sub>O<br>
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-autoclave<br>
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Note: also possibility to use 1X PBS tablets (one tablet per 200ml)<br>
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</html>
</html>

Latest revision as of 14:23, 14 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

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