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From 2011.igem.org

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.

4th of August

In order to make the stock solutions of the Salkowski reagent that was used for the PC method we mixed 100ml of 7.9M sulphuric acid with 1.49g of FeCl2.(4H2O). We called this mixture R1.

In order to make the stock solutions of the Salkowski reagent that was used for the S2/1 method we mixed 100ml of 10.8M sulphuric acid with 0.55g of FeCl2.(4H2O). We called this mixture R2.

The PC method allows us to measure Auxin levels of 0.2μg/ml to 20 μg/ml and is far more specific for IAA rather than other indoles. To perform this experiment:
-Mix 500 μl of Trp supplemented LB broth (1 mg/ml) with 500 μl of R1.
-Leave in dark at room temperature for 30 minutes.
-Measure OD at 533nm.

The S2/1 method allows us to measure a range from 2 μg/ml to 200 μg/ml but is less specific for IAA. In order to perform this experiment:
-Mix 500 μl of Trp supplemented LB broth (1 mg/ml) with 1000 μl of R1.
-Leave in dark at room temperature for 30 minutes.
-Measure OD at 554nm.