IGEM:IMPERIAL/2007/Experimental Design/Phase2/Results 1.1: Difference between revisions

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==Results Summary==
= Fluorescence Dependence on Media Volume=
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===<div style="color:darkblue; font-size: 140%;">GFP with variable water volumes</div>===
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==Aims==
To investigate the fluorescence dependence of varying media volumes to no. of moles of GFP. This is to determine the need for the maintenance of a constant volume throughout subsequent experiments, as with making GFP dilutions for the calibration curve.
 
 
==Materials and Methods==
Refer to protocols page.
 
==Results==
[[Image:IC2007 Experimental Design GFP Dilution in water.PNG|thumb|left|800px| Fig.1: Fluorescence of varying media volume]]
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This purpose of this experiment was to investigate whether the same number of moles of GFP, in different water volumes gave a different fluorescence reading or not. Depending on the results, we would see whether we needed or not to maintain constant volumes when making GFP dilutions for our calibration curve.


We therefore added 4 different volumes of water in 4 wells and in each well we added the same amount of GFP (from the same stock soluton). Two wells were also used to contain just GFP(+ve) and just water(-ve) as a controls. The results are shown below:
Interestingly, the addition of more media (water) increased the fluorescence readings of the samples to a significant extent (Fig.1).
 
 
==Discussion==
Fig.1 suggests that fluorescence does depend on the wolume of media to which the substrate is diluted in, albeit in a proportionate way. While one would expect the opposite to occur, the results suggest that volume of media, and hence the maintenance of the same volume through subsequent experiments, is vital for an accurate reading. Indeed this also implicitly implies the importance of minimizing evaporation in our media.


[[Image:IC2007 Experimental Design GFP Dilution in water.PNG|thumb|left|800px| Results of testing pLux in vivo with a consntant AHL concentration]]


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==Conclusion==
On the graph on the right you can see the fluorescence reading of the pLux assembly 7 hours after it has been induced with AHL. The readings of pLux are comparable to the diluted GFP solution and leads us to conclude that GFP has been expressed. Thus the contruct works as expected in vivo. Further tests will need to be conducted to reveal its sensitivity to AHL.
* Constant volume must be maintained for all experiments.
* Evaporation (reducing media volume) may play a huge role in experimental readings.

Latest revision as of 04:04, 16 October 2007

Fluorescence Dependence on Media Volume

Aims

To investigate the fluorescence dependence of varying media volumes to no. of moles of GFP. This is to determine the need for the maintenance of a constant volume throughout subsequent experiments, as with making GFP dilutions for the calibration curve.


Materials and Methods

Refer to protocols page.

Results

Fig.1: Fluorescence of varying media volume


Interestingly, the addition of more media (water) increased the fluorescence readings of the samples to a significant extent (Fig.1).


Discussion

Fig.1 suggests that fluorescence does depend on the wolume of media to which the substrate is diluted in, albeit in a proportionate way. While one would expect the opposite to occur, the results suggest that volume of media, and hence the maintenance of the same volume through subsequent experiments, is vital for an accurate reading. Indeed this also implicitly implies the importance of minimizing evaporation in our media.


Conclusion

  • Constant volume must be maintained for all experiments.
  • Evaporation (reducing media volume) may play a huge role in experimental readings.
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