IGEM:IMPERIAL/2007/Experimental Design/Phase2/Protocol 1.0: Difference between revisions

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'''Aims:'''<br>
'''Aims:'''<br>  
*The aim of the calibration curve is to determine a relationship between molecules of GFP in our cell extract and fluorescence. It should be noted that throughout our phase 2 experiments we will be maintaining the volume of cell extract used.
*The purified GFP that we wish to use for our calrbration curve is stored in a solution of buffer. Using this GFP there are several approachs to construct a calubration curve, and the purpose of this experiment is to validate one of these method. The approaches are:<br>
 
 
''Experiment 1:
<br>
To use the [GFP] solution and create several dilutions using distilled water. Assumes that [GFP] has the same fluorescence in water and Cell extract.
<br>
''Experiment 2:
<br>
To maintain the constant volume of cell extract used for phase 2 and add a [GFP] solution. Assumes that the the total volume of sample has no affect on fluorescence as long as moles of GFP are constant.
<br>
''Experiment 3:
<br>
To use less volume than the constant cell extract used for phase 2 and add a [GFP] solution, this will bring the total volume to that used for phase 2 experiments. Assumes that varing volumes of cell extract volume has no affect on fluorescence i.e. minimal flourescence absorbance from cell extract.
*We need to test these three methods to see which is the best for constructing a calibration curve
*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
#Experiment 3 - Test the same [GFP] in different cell extract volumes but keeping total volume the same.
'''Status:'''
'''Status:'''
*To be carries out on 20/08/2007
*To be carries out on 20/08/2007
Line 35: Line 23:
====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 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
#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 200ul 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 300ul <br>'''Dilution 3'''- Add 500ul<br>'''Make sure that the cell extract has completly melted before adding to eppendorf tubes.
''Experiment 1''
<br>
#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.
<br>
''Experiment 2''
<br>
#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:<br>'''2.a''' - Add 100ul <br>'''2.b'''- Add 300ul <br>'''2.c'''- Add 500ul<br>'''  
#Place these three eppendorf tubes in a rack and put to the side until loading up the plate <br><br>
#Place these three eppendorf tubes in a rack and put to the side until loading up the plate <br><br>
#Now prepare for ''experiment 2''. Label 2x eppendorf tubes as follows; '''x200''', '''x400'''. These correspond to the different dilutions used.
#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 distilled water. 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 distilled water. 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 distilled water. 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 distilled water. This solution is now x400 fold dilution of the unkown [GFP] solution. Place this tube in the rack.

Revision as of 10:18, 19 August 2007

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 300ul
    2.c- Add 500ul
  2. Place these three eppendorf tubes in a rack and put to the side until loading up the plate

  3. Now prepare for experiment 2. Label 2x eppendorf tubes as follows; x200, x400. These correspond to the different dilutions used.


  1. To the x200 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.
  2. To the x400 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 None
A2 Dilution 1 100 None
A3 Dilution 2 200 None
A4 Dilution 2 200 None
A5 Dilution 3 300 None
A6 Dilution 3 300 None
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

Dependece

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