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Team:Imperial College London/Project/Auxin/Protocols
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<b>New protocol:</b><br> | <b>New protocol:</b><br> | ||
50μg/ml is the recommended concentration. We will use 35-40μg/ml of kanamycin to speed up growth.</html> | 50μg/ml is the recommended concentration. We will use 35-40μg/ml of kanamycin to speed up growth.</html> | ||
| + | <html> | ||
| + | <h2>28th of July</h2> | ||
| + | Transformation of cells with 6, 7 and 8:<br><br> | ||
| + | -Let competent cell strain 5α thaw for around 10 minutes on ice.<br> | ||
| + | -Add 2-3μl of DNA.<br> | ||
| + | -Leave on ice for 20-25 minutes.<br> | ||
| + | -Heat shock cells at 42°C for 45 seconds.<br> | ||
| + | -Leave on ice for 10 minutes.<br> | ||
| + | -Add 500μl of LB broth.<br> | ||
| + | -Incubate for 1 hour at 37°C.<br> | ||
| + | -Centrifuge for 1 minute.<br> | ||
| + | -Remove 100μl off the top of the eppendorf tube. Pour out the rest making sure that the pellet remains in the eppendorf tube.<br> | ||
| + | -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 the rest of the sample to a second chloramphenicol agar plate.</html> | ||
Revision as of 13:28, 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.
