Team:Imperial College London/Extras/Protocols/Chemotaxis

From 2011.igem.org

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<h1>Chemotaxis Lab Protocols</h1>
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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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<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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- 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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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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<h2>2nd of August</h2>
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Preparation of semi-solid agar used for qualitative experiments, the recipe is for total volume of 1 litre: <br>
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- 5g NaCl<br>
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- 10g tryptone<br>
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- 2g D-glucose<br>
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- 3g agar<br>
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- 1000ml H<sub>2</sub>O<br>
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- autoclave<br>
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- before making plates, cool down semi-solid agar to 50<sup>o</sup>C and add required amount of antibiotics
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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