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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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| - | -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><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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| - | </html>
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