Team:UNIPV-Pavia/Methods Materials/Absorbance
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As regards the other ambients, we observed a longer exponential phase (about 6 hours), with a final O.D. value of 0.46 for the flask and 0.51 for the falcon tube, and a doubling time of 55 minutes for both ambients. | As regards the other ambients, we observed a longer exponential phase (about 6 hours), with a final O.D. value of 0.46 for the flask and 0.51 for the falcon tube, and a doubling time of 55 minutes for both ambients. | ||
- | This analysis allows us to individuate another important protocol to control the exponential phase, to have the best gene expression. The starting point is protocol n.1, described in , the new step is performing a dilution after the 2/3-hours incubation till reaching an O.D. value of about 0.02: wells filled with this colture will be in the exponential phase for about two hours after the incubation. | + | This analysis allows us to individuate another important protocol to control the exponential phase, to have the best gene expression. The starting point is protocol n.1, described in [[Team:UNIPV-Pavia/Methods_Materials/Absorbance#Different protocols for different growth curves|Different protocols for different growth curves]], the new step is performing a dilution after the 2/3-hours incubation till reaching an O.D. value of about 0.02: wells filled with this colture will be in the exponential phase for about two hours after the incubation. |
Revision as of 21:04, 18 October 2009
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Measurements - Absorbance |
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Absorbance and water dispensationWe find no relevant differences between the measures before and after the dispensation of different volumes of water in the well, both for simple liquid growth medium and bacterial coltures. You can find more informations about the experiment at [http://aimed11.unipv.it/iGEM2009 Download Protocol] - Test 17/10/09.
Absorbance and volumeThe proportionality between absorbance (OD600) measurement and volume of colture in the well was verified. By fitting a linear regression model on the experimental data we estimate a coefficient of proportionality of 8.27e-4 for bacterial coltures and of 1.38e-4 for LB+Amp medium. You can find more informations about the experiments at [http://aimed11.unipv.it/iGEM2009 Download Protocol] - Test n.9bis, 10/10/09 (bacterial colture), and Test 17/10/09 (LB medium).
Absorbance and dilutions in liquid growth mediumThe proportionality between absorbance measurement (OD600) and colture's dilutions was verified, confirming the possibility of using OD600 to measure the bacterial quantity in the well, regardless of the total volume present in the well. This experiment was done in two different growth mediums, LB and M9, and in three different total volumes in the well, 100μl, 200μl and 300μl. By fitting a linear regression model on the experimental data we estimate a coefficient of proportionality of 0.0018 for LB medium and 0.0035 of for M9. You can find more informations about the experiments at [http://aimed11.unipv.it/iGEM2009 Download Protocol] - Test n.20, 13/08/09 and Test n.20 "M9", 28/08/09.
Different protocols for different growth curvesAs our first step we wanted to individuate a proper protocol to have reproducible growth curves for the coltures incubated inside the microplate reader. We compared three different solutions, as you can see in the figures below. Protocol 1:
Protocol 2:
Protocol 3:
You can find more informations about the experiment at [http://aimed11.unipv.it/iGEM2009 Download Protocol] - Test n.5, 18/06/09.
On the other hand, protocol n.1 ensures a perfect reproducibility of the curves coming from the same falcon tube. Clearly coltures from different falcon tubes evolve in different way. For all these reasons we decide to use this protocol in our experiments.
You can find more informations about the experiment at [http://aimed11.unipv.it/iGEM2009 Download Protocol] - Test n.6, 27/06/09.
Growth ambients in comparisonA 10-hours experiment was made to compare the growth in the microplate with those in typical growth ambients like falcon tube and flask, especially in the exponential phase. The goal was the estimation of important parameters that characterize growth curves, as duration and evolution of all the phases, and doubling time in exponential phase. As you can see in the figures below, growth inside the microplate is well comparable to the other two till the end of its exponential phase (4 hour, estimation done on the logarithm of the data), then slowes down, becoming almost linear, to reach O.D. values lower than the others (0.4). At the end of the exponential phase the O.D. measurement is 0.14 and the doubling time estimate is 40 minutes (referring to the logistic growth model): parameters consistent with those present in literature. As regards the other ambients, we observed a longer exponential phase (about 6 hours), with a final O.D. value of 0.46 for the flask and 0.51 for the falcon tube, and a doubling time of 55 minutes for both ambients. This analysis allows us to individuate another important protocol to control the exponential phase, to have the best gene expression. The starting point is protocol n.1, described in Different protocols for different growth curves, the new step is performing a dilution after the 2/3-hours incubation till reaching an O.D. value of about 0.02: wells filled with this colture will be in the exponential phase for about two hours after the incubation.
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