Team:UNIPV-Pavia/Methods Materials/Absorbance

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Revision as of 11:08, 17 October 2009

Measurements - Absorbance

Absorbance and water dispensation

We find no relevant differences between the measures before and after the dispensation of different volumes of water in the well, both for bacterial coltures and simple liquid growth medium.

You can find more informations about the experiment at [http://aimed11.unipv.it/iGEM2009 Download Protocol] - Test n.9bis, 10/10/09.

You can find more informations about the experiment at [http://aimed11.unipv.it/iGEM2009 Download Protocol] - Test 17/10/09.

Absorbance and water dispensation

Absorbance and volume

The 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.

You can find more informations about the experiment at [http://aimed11.unipv.it/iGEM2009 Download Protocol] - Test n.9bis, 10/10/09.

Absorbance and volume

Absorbance and dilutions in liquid growth medium

The 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.

Absorbance and colture's dilutions in LB

Absorbance and colture's dilutions in M9

Different protocols for different growth curves

As 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:

overnight colture incubation from glycerol stock (8 μl) in 5ml LB+Amp 37°C 220 rpm;
dilution 1:1000;
incubation 37°C 220 rpm for about 2/3 hours;
use this colture to fill the plate.

Protocol 2:

pick a colony from a streaked plate with single colonies;
infect the microplate well, previously filled with 200 μl of growth medium.

Protocol 3:

overnight colture incubation from glycerol stock (8 μl) in 5ml LB+Amp 37°C 220 rpm;
dilution 1:100;
incubation 37°C 220 rpm for about 2/3 hours;
infect the microplate well, previously filled with 200 μl of growth medium, with a sterile pipette tip poured in the grown colture.

Growth Protocols n.1 and n.2

Growth Protocol n.3

Growth ambients in comparison

Growth ambients in comparison, exponential phase

@@ Line 73: / Line 73: @@
 ''Protocol 1'':
-*overnight colture incubation in 5ml LB+Amp 37°C 220 rpm;
+*overnight colture incubation from glycerol stock (8 μl) in 5ml LB+Amp 37°C 220 rpm;
-*dilution 1:100;
+*dilution 1:1000;
-*incubation 37°C 220 rpm for about 4 hours.
+*incubation 37°C 220 rpm for about 2/3 hours;
+*use this colture to fill the plate.
 ''Protocol 2'':
@@ Line 82: / Line 83: @@
 ''Protocol 3'':
-*
+*overnight colture incubation from glycerol stock (8 μl) in 5ml LB+Amp 37°C 220 rpm;
+*dilution 1:100;
+*incubation 37°C 220 rpm for about 2/3 hours;
+*infect the microplate well, previously filled with 200 μl of growth medium, with a sterile pipette tip poured in the grown colture.
 [[Image:unipv_asb_protocols_12.jpg| 500px|Growth Protocols n.1 and n.2|thumb|center|Growth Protocols n.1 and n.2]]
 [[Image:unipv_asb_protoco3.jpg| 500px|Growth Protocol n.3|thumb|center|Growth Protocol n.3]]