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Team:Imperial College London/Project/Chemotaxis/Protocols
From 2011.igem.org
(Difference between revisions)
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-Re-suspend the cells in the 100μl LB broth solution that was removed in the previous step. | -Re-suspend the cells in the 100μl LB broth solution that was removed in the previous step. | ||
-Add 5μl on a chloramphenicol agar plate (concentration of 35μg/ml).<br> | -Add 5μl on a chloramphenicol agar plate (concentration of 35μg/ml).<br> | ||
| - | -Add the rest of the sample to a second chloramphenicol agar plate.</html> | + | -Add the rest of the sample to a second chloramphenicol agar plate.<br></html> |
| + | <html><h2>28th of July</h2><br> | ||
| + | To make tryptone broth (bacterial growth before chemotaxis assays)for total volume of 1L:<br> | ||
| + | -10g tryptone<br> | ||
| + | -1000ml of 1X PBS<br> | ||
| + | -autoclave<br> | ||
| + | -1.14g kanamycin<br> | ||
| + | To make 1X PBS (phosphate buffer saline):<br> | ||
| + | -in 800ml of distilled H<sub>2</sub>O<br> | ||
| + | -8g of NaCl<br> | ||
| + | -0.2g of KCl<br> | ||
| + | -1.44g of Na<sub>2</sub>HPO<sub>4</sub><br> | ||
| + | -0.24g of KH<sub>2</sub>PO<sub>4</sub><br> | ||
| + | -adjust pH to 7.4<br> | ||
| + | -adjust volume 1L with additional distilled H<sub>2</sub>O<br> | ||
| + | -autoclave<br> | ||
| + | Note: also possibility to use 1X PBS tablets (one tablet per 200ml)<br> | ||
| + | </html> | ||
Revision as of 14:47, 28 July 2011
27th of July
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:-No innoculation
-Kanamycin concentration in LB broth was too high. We had used 85μg/ml.
New protocol:
50μg/ml is the recommended concentration. We will use 35-40μg/ml of kanamycin to speed up growth.
28th of July
Transformation of cells with 6, 7 and 8:-Let competent cell strain 5α thaw for around 10 minutes on ice.
-Add 2-3μl of DNA.
-Leave on ice for 20-25 minutes.
-Heat shock cells at 42°C for 45 seconds.
-Leave on ice for 10 minutes.
-Add 500μl of LB broth.
-Incubate for 1 hour at 37°C.
-Centrifuge for 1 minute.
-Remove 100μl off the top of the eppendorf tube. Pour out the rest making sure that the pellet remains in the eppendorf tube.
-Re-suspend the cells in the 100μl LB broth solution that was removed in the previous step. -Add 5μl on a chloramphenicol agar plate (concentration of 35μg/ml).
-Add the rest of the sample to a second chloramphenicol agar plate.
28th of July
To make tryptone broth (bacterial growth before chemotaxis assays)for total volume of 1L:
-10g tryptone
-1000ml of 1X PBS
-autoclave
-1.14g kanamycin
To make 1X PBS (phosphate buffer saline):
-in 800ml of distilled H2O
-8g of NaCl
-0.2g of KCl
-1.44g of Na2HPO4
-0.24g of KH2PO4
-adjust pH to 7.4
-adjust volume 1L with additional distilled H2O
-autoclave
Note: also possibility to use 1X PBS tablets (one tablet per 200ml)
