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Team:Imperial College London/Project/Auxin/Protocols
From 2011.igem.org
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-Centrifuge for 1 minute.<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> | -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. | + | -Re-suspend the cells in the 100μl LB broth solution that was removed in the previous step.<br> |
-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.</html> | ||
Revision as of 09:08, 29 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.
