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.
2. To use less cell extract that we will be using in the phase 2 experiments and add to this varying [GFP] solutions. This time the total volume of this solution will be the same as that for the phase 2 experiments. We need to test the affect how varying volumes of cell extract will affect the fluorescence. Dependence on cell extract volume used in a constant total volume

Status:

Equipment

• Fluorometer
• 6x Eppendorf tubes
• Eppendorf Rack
• Pen

Reagents

• 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

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 add 10ul of unknown [GFP] and place in a the eppendorf tube. To this then add 990ul of distilled water, first use a p1000 to remove 900ul and then add 90ul with a p200. Return the sample of unknown [GFP] back to fridge
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 2x 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.

Loading the Plate

1. 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.

Well Solution Added Volume (ul) A1 Dilution 1 200 A2 Dilution 1 200 A2 Dilution 2 200 A3 Dilution 2 200 A4 Dilution 3 200 A5 Dilution 3 200 C1 pTet Commercial E.coli extract C1 pTet Commercial E.coli extract C1 pTet Commercial E.coli extract C1 pTet Commercial E.coli extract C1 pTet Commercial E.coli extract C1 pTet Commercial E.coli extract E1 pcI Commercial E.coli extract E2 pcI Commercial E.coli extract