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Team:Imperial College London/Extras/Protocols/Auxin
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Revision as of 14:26, 2 August 2011
Auxin Lab Protocols
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.
