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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

• Raw Data excel file

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 increase 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.