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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.

Salkowski reagent revised

- 0.811g of anhydrous FeCl₃ is dissolved in 10ml H₂O to obtain 0.5M solution
- add 1ml of FeCl₃ 0.5M solution to 50ml of 35% HClO₄
- store at room temperature in absence of sunlight
- To a 1.0-ml aliquot/supernatant add 2.0 ml of FeCl3-HCl04 reagent
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.

9th of August

To prepare the E.Coli, media and soil for the survivability experiement:
-Make filter paper discs for soaking the media in.
-Transform E.Coli K-12 5α, with a high-copy plasmid containing a GFP expression.
-Innoculate colonies from the transformants into a 4ml LB media and incubate at 37°C and 225 rpm for a period of 5 hours.
-Place 1g of soil into eppendorf tubes 60 times. Autoclave 30 tubes.
-Pour bacteria into two 2ml eppendorf tubes, centrifuge and empty broth, ensuring pellet remains in the eppendorf tube.
-Re-suspend in 1ml LB.
-Place 10µl each on 60 filter paper discs, and place one in each eppendorf tube with soil in it.
-Add Kanamycin to 400 ml of LB to give concentration of 35μg/ml.
-Add 1 ml of the mixture to 60 separate eppendorf tubes.
-Freeze Media.

Taking Results:
-Remove 6 media eppendorfs from freezer, place in 37°C to melt.
-Remove Filter paper disc from soil tubes and put one each into melted media. Try to shake off the soil from the discs. Red is sterile, blue is non-sterile.
-Incubate at 13:00 at 37°C and 225 rpm for 4 hours.
-Record OD600 of tubes.

22nd of August

To rehydrate synthesised DNA:
- Centrifuge for 4 minutes to ensure DNA is at the bottom of the tube
- Consult the Synthesis Report to work out how much water to add - multiply the yield in nmol by 10, and that value is the volume of water in µl
- Gently pipette up and down, then leave for 5 minutes
- Vortex, pipette up and down, and then leave for a further 5 minutes
- Take 10µl of this DNA solution and add it to 90µl of pure water to make a working solution