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

UNDER CONSTRUCTION

Aims:

• 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:

1. Experiment 1 - Test the difference in GFP dilutions in water and cell extract
2. Experiment 2 - Test the same moles of GFP in different total volumes
3. Experiment 3 - Test the same [GFP] in different cell extract volumes but keeping total volume the same.

Status:

• To be carries out on 20/08/2007

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

Experiment 1

1. 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.

Experiment 2

1. 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
   
2. Place these three eppendorf tubes in a rack and put to the side until loading up the plate
   
   

Experiment 3

1. Now prepare for experiment 3. Label 2x eppendorf tubes as follows; "3.a", "3.b.
2. To the 3.a tube add 200ul of the x100 solution, then add 200ul of the distilled water. This solution is now x200 fold dilution of the unkown [GFP] solution.Place this tube in the rack.
3. To the 3.b tube remove 200ul of the x200 solution, then add 200ul of distilled water. 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 GFP Solution Added Volume of GFP Solution Added(ul) Volume of Home made Cell extract added (ul) A1 Dilution 1 100 100 A2 Dilution 1 100 100 A3 Dilution 2 200 100 A4 Dilution 2 200 100 A5 Dilution 3 300 100 A6 Dilution 3 300 100 C1 x100 50 150 C2 x100 50 150 C3 x200 100 100 C4 x200 100 100 C5 x400 200 None C5 x400 200 None E1 None - 100 E2 None - 100