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| | + | {{:Team:Imperial_College_London/Templates/Header}} |
| | + | {{:Team:Imperial_College_London/Templates/Protocols}} |
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| | <html> | | <html> |
| - | <h1>Chemotaxis Lab Protocols</h1> | + | <body> |
| - | <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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| - | -No innoculation<br>
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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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| - | 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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| - | 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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| - | - Add 2-3μl of DNA.<br>
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| - | - Leave on ice for 20-25 minutes.<br>
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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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| - | - Add 500μl of LB broth.<br>
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| - | - Incubate for 1 hour at 37°C.<br>
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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> | + | <h1>Phyto-Route</h1> |
| - | <b>Antibiotics</b>:<br>
| + | <h2>Tryptone Broth</h2> |
| - | Four different antibiotics (kanamycin, chloramphenicol, ampicillin & tetracycline) have been used during the course of the project. They have been used in following working concentrations, unless stated otherwise:<br>
| + | <hr style="color:#225323;"/> |
| - | - Kanamycin - 35µg/ml<br> | + | <h2>PBS</h2> |
| - | - Chloramphenicol – 35µg/ml<br>
| + | <hr style="color:#225323;"/> |
| - | - Tetracycline – 35 µg/ml<br>
| + | <h2>Motility Medium</h2> |
| - | - Ampicillin – 100 µg/ml<br><br>
| + | <hr style="color:#225323;"/> |
| - | <b>Tryptone broth</b><br>
| + | <h2>Preparation</h2> |
| - | To make bacteria develop flagella they are grown in the tryptone broth instead of LB broth. This is recipe for total volume of 1L:<br>
| + | <hr style="color:#225323;"/> |
| - | - 10g tryptone<br>
| + | <h2>Agar Plugin</h2> |
| - | - 1000ml of 1X PBS<br>
| + | <hr style="color:#225323;"/> |
| - | - autoclave<br>
| + | <h2>Semi-solid Agar</h2> |
| - | - add required amount of antibiotics<br>
| + | <hr style="color:#225323;"/> |
| - | <br>
| + | <h2>Capillary Assay</h2> |
| - | To make 1X PBS (phosphate buffer saline):<br>
| + | <hr style="color:#225323;"/> |
| - | -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>
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| - | <h2>2nd of August</h2><br> | + | |
| - | <b>Semi - solid agar</b><br> | + | |
| - | In chemotaxis assays semi-solid agar is used as it allows greater diffusion of molecules and allows movement of bacteria within agar. This is recipe 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 in water-bath and add required amount of antibiotics<br>
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| - | <h2>5th of August</h2> | + | |
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| | + | </body> |
| | </html> | | </html> |
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