IGEM:IMPERIAL/2007/Experimental Design/Phase2/Protocol 1.0: Difference between revisions
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====Protocol==== | ====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. | *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 an eppendorf tube '''x100''' then to this | #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 | ||
#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:<br>Dilution 1 - Add 100ul <br>Diltuion 2- Add 200ul <br>Dilution 3- Add 300ul<br>'''Make sure that the cell extract has completly melted before adding to eppendorf tubes. | #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:<br>'''Dilution 1''' - Add 100ul <br>'''Diltuion 2'''- Add 200ul <br>'''Dilution 3'''- Add 300ul<br>'''Make sure that the cell extract has completly melted before adding to eppendorf tubes. | ||
#Place these three eppendorf tubes in a rack and put to the side until loading up the plate | #Place these three eppendorf tubes in a rack and put to the side until loading up the plate | ||
#Now prepare for experiment 2. Label | #Now prepare for experiment 2. Label 2x eppendorf tubes as follows; '''x200''', '''x400'''. These correspond to the different dilutions used. | ||
#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. | #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. | ||
#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. | #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. | ||
<br> | <br> | ||
'''Loading 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. | #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. |
Revision as of 08:22, 19 August 2007
Experiment 1
Aims:
- To validate which of the following approaches should be used for calibration curve:
- 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.
- 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
Reagents
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.
- 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
- 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. - Place these three eppendorf tubes in a rack and put to the side until loading up the plate
- Now prepare for experiment 2. Label 2x eppendorf tubes as follows; x200, x400. These correspond to the different dilutions used.
- 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.
- 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
- 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.