Imperial/Wet Lab/Results/CBD2.1

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__NOTOC__
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=Test for operation at different temperatures for pTet-GFPmut3b=
 
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== [http://partsregistry.org/Part:BBa_I13522 '''pTet-GFP''']==
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=Wet Lab: ResultsTest for optimum packaging and sampling techniques=
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==Aim==
==Aim==
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The aim of these experiments was to firgure out the operating range of the pTet-GFP construct in-vitro. The temperatures the construct was tested in are: 8&deg;C, 10&deg;C, 20&deg;C and 37&deg;C.
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*Usage of Construct [http://partsregistry.org/Part:BBa_I13522 '''pTet-GFP''']
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<br>'''31-08-2007'''
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*Determine an optimum packaging technique for the experiments, that minimises the evaporation of the reaction mixture.
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*Determine a suitable sampling technique which disrupts the reaction to a very minimum
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<br>'''07-10-2007'''
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==='''10&deg;C'''===  
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==Materials and Methods==
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pTet was tested at 10<sup>o</sup>C for 4 hours. The results show no expression of pTet-GFP. After this experiment we took the samples out and put them at room temperature over night, this is to see if the low temperature permanently inactivate the in vitro chassis or DNA.
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See [[Imperial/Wet_Lab/Protocols/CBD2.1|Protocols]] page
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==Results==
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{| border="2" style="background:#ABCDEF;"  
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{|align="center"
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| [[Media:PTet-in-vitro-10oC.xls|Complete set of results and raw data ]]
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| width="600px"|<br>[[Image:IC 2007 packaging.PNG|thumb|600px|Fig.1.1: '''pTet-GFPmut3b''' - fluorescence output for various packaging and sampling techniques, over a period of 6 hours]]  
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==='''20&deg;C'''===
 
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pTet was tested at 20&deg;C for two days.
 
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{| border="2" style="background:#ABCDEF;"
 
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| [[Media:IC 2007 Ptet 20oC-1.xlsPtet 20oC-1.xls|Complete set of results and raw data ]]
 
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==='''37&deg;C'''===
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'''Controls:'''
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pTet was tested at 37&deg;C for two days and a staggered experiment was carried out to minimise the missed measurements over night.  
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*Negative Control - 96 well plate with singl layered tape and no pipetting (same experimental conditions as have them usually)
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{| border="2" style="background:#ABCDEF;"
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'''Constants:'''
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| [[Media:Graph 37oC.xls|Complete set of results and raw data ]]
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*Temperature -  37&deg;C
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*Volume of Cells sampled - 60&micro;l
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'''Raw Data'''
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*[[Media:IC 2007 CBD Packaging.xls|Raw Data excel file]]
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==Discussion==
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It can be seen from the results that the fluorescence is the highest for the plate with multiple layers of tape. Thus putting extra tape on the wells changes the fluorescence of the system. As it has been seen before, evaporation of samples results in greater fluorescence, thus there is more evaporation in the multi-layered plate.
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For the pipetting sample, two readings were taken for each data point. One before and one after pipetting the mixture. It can be seen that pipetting disrupts the reaction as fluorescence after pipetting increased to a very high value.
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The PCR method seems to have the least fluorescnce out of all the samples. As PCR method requires pipetting and centrifuging (not tested here), there will be a lot of disruption in the reaction.
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==Conclusion==
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It can be concluded from the above results that it is not possible to carry out experiments over a time period greater than  one day (about 9 hours of lab). This is as it is not possible to reduce evaporation so it is not signifcant over a few days. Having a stock solution of the reaction mixture is also not feasible as the cell extract is very expensive.

Latest revision as of 03:00, 27 October 2007



Wet Lab: ResultsTest for optimum packaging and sampling techniques

Aim

  • Usage of Construct [http://partsregistry.org/Part:BBa_I13522 pTet-GFP]
  • Determine an optimum packaging technique for the experiments, that minimises the evaporation of the reaction mixture.
  • Determine a suitable sampling technique which disrupts the reaction to a very minimum


07-10-2007

Materials and Methods

See Protocols page

Results


Fig.1.1: pTet-GFPmut3b - fluorescence output for various packaging and sampling techniques, over a period of 6 hours


Controls:

  • Negative Control - 96 well plate with singl layered tape and no pipetting (same experimental conditions as have them usually)

Constants:

  • Temperature - 37°C
  • Volume of Cells sampled - 60µl


Raw Data

Discussion

It can be seen from the results that the fluorescence is the highest for the plate with multiple layers of tape. Thus putting extra tape on the wells changes the fluorescence of the system. As it has been seen before, evaporation of samples results in greater fluorescence, thus there is more evaporation in the multi-layered plate.

For the pipetting sample, two readings were taken for each data point. One before and one after pipetting the mixture. It can be seen that pipetting disrupts the reaction as fluorescence after pipetting increased to a very high value.

The PCR method seems to have the least fluorescnce out of all the samples. As PCR method requires pipetting and centrifuging (not tested here), there will be a lot of disruption in the reaction.

Conclusion

It can be concluded from the above results that it is not possible to carry out experiments over a time period greater than one day (about 9 hours of lab). This is as it is not possible to reduce evaporation so it is not signifcant over a few days. Having a stock solution of the reaction mixture is also not feasible as the cell extract is very expensive.

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