IGEM:IMPERIAL/2007/Experimental Design/Phase2/Protocol 1.0

Experiment 1

Aims:

• To validate which of the following approaches should be used for calibration curve:

1. To maintain a constant amount of cell extract used for phase 2 and add a set volume of varying [GFP] in solution. However, this varies the total volume of sample measured and so we need to test if constant moles of GFP have the same fluorescence in varying volumes. Dependence on total volume , with constant phase 2 volume of cell extract used.

Status:

Protocol

• 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.

1. First perform a 100 fold dilution on the unknown[GFP] solution. Label an eppendorf tube x100 then to this remove 10ul of unknown [GFP] and place in a 1.5ml eppendorf tube. To this then add 990ul of distilled water, first use a p1000 to remove 900ul and then add 90ul with a p200. This solution then should be put on ice.
2. Now we can prepare experiment 1. First label three 1.5ml eppendorf tubes with dilution 1, dilution 2 and dilution 3. Then remove 100ul of the x100 dilution into each eppendorf tube. Now add the following volumes of home made cell extract to the following tubes:
   Dilution 1 - Add 100ul
   Diltuion 2- Add 200ul
   Dilution 3- Add 300ul
   Make sure that the cell extract has completly melted before adding to eppendorf tubes.
3. Place these three eppendorf tubes in a rack and put to the side until loading up the plate
4. Now prepare for experiment 2. Label ...eppendorf tubes as follows; x200, x400. These correspond to the different dilutions used.
5. To the x200 tube add 200ul of the x100 solution, then add 200ul of the home made cell extract solution. This solution is now x200 fold dilution of the unkown [GFP] solution.Place this tube in the rack.
6. To the x400 tube remove 200ul of the x200 solution, then add 200ul of home made cell extract solution. This solution is now x400 fold dilution of the unkown [GFP] solution. Place this tube in the rack.

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