IGEM:IMPERIAL/2007/Experimental Design/Phase2/Protocol 1.1
- We need to test these three methods to see which is the best for constructing a calibration curve
- Three tests will be carried out to determine which is the best approach:
- Experiment 1 - Test the difference in GFP dilutions in water and cell extract
- Experiment 2 - Test the same moles of GFP in different total volumes
- To be carries out on 20/08/2007
- 8x Eppendorf tubes
- Eppendorf Rack
- 2ml Tube
- 10x150ul of Home made Cell extract from freezer. (x10 allows for spares)
- 1x Solution of unknown [GFP] from fridge. It is a clear eppendorf tube
- Distilled water
- (Note: The EGFP and GFP-S65T variants exhibit a reduced range of pH stability. For these variants, fluorescence is stable between pH 7.0 and pH 11.5, drops sharply above pH 11.5, and decreases between pH 7.0 and pH 4.5, retaining about 50% of fluorescence at pH 6.0 (Patterson et al., 1997; Ward, pers. comm.))
- We do not yet have our sample of purified GFP and so we are using a sample of unknown [GFP] in solution from last years Imperials iGEM team. We can use this sample because we do not need to know the exact concentration of GFP. We have already tested this GFP solution and have found a dilution of 100 fold to give a strong reading in the fluorometer.
- First perform a 100 fold dilution on the unknown[GFP] solution. Label a 2ml tube x100 then to this add 20ul of unknown [GFP] and place in a 2ml tube. To this then add 1980ul of distilled water, first use a p1000 to remove 1000ul then 980ul. Return the sample of unknown [GFP] back to fridge
- Now we can prepare for experiment 1. Collect 2x Eppendorf tubes and label "1.a" and "1.b.". To each of these tubes add 200ul of x100 GFP solution. Then to tube 1.a Add 200ul of home made Cell Extract and mix thoroughly. Then to tube 1.b. add 200ul of distiled water and mix thoroughly. Return tubes to the rack.
- Now we can prepare experiment 2. First label three 1.5ml eppendorf tubes with 2.a, 2.b and 2.c. Then remove 200ul of the x100 dilution into each eppendorf tube. Now add the following volumes of distilled water to the following tubes:
2.a - Add 100ul
2.b- Add 200ul
2.c- Add 400ul
- Place these three eppendorf tubes in a rack and put to the side until loading up the plate
Loading the Plate
- Follow the schematic for the plate and begin by loading the in vitro expression system into the correct wells. Before loading in the samples vortex the tubes for a few seconds to mix the solution.