Biology Reference
In-Depth Information
colonies and then placed into media in a 96-well plate with some agitation. Each well
contains 100
l of broth and antibiotics as appropriate. This plate is incubated in
a sealed plastic bag (to prevent evaporation) overnight at 37
C with shaking.
The experimental plate is prepared by transferring 1
m
l of each overnight culture
using the multichannel pipette to the wells of the experimental plate containing
100
m
l of the desired culture medium. All wells of the experimental plate should con-
tain culture medium even though the outer wells are not inoculated with any strains.
These wells are used for background fluorescence readings, as controls to indicate
that cross-well contamination has not occurred and to maintain humidity and reduce
evaporation in the inner wells.
A typical protocol for an experiment starting with frozen stocks in the 96-well
storage plate is as follows:
m
70
C freezer and allow to thaw for
1.
Remove the storage plate from the
5 min.
2.
Generate a grid of fresh colonies on a square culture dish containing LB agar with
appropriate antibiotic additions, using a sterilized Boekel 96-pin replicator.
3.
Generate overnight cultures (100
m
l) by inoculating a 96-well plate (Falcon
353072) from the grid of strains using a multichannel pipette with disposable tips.
This 96-well plate is placed in a plastic bag to prevent evaporation and cultivated
overnight at 37
C under constant shaking of 1050 rpm in a Heidolph titramax
1000 (Heidolph UK, Essex, UK) incubator.
4.
The experimental plate is prepared by transferring 1
l of the overnight culture to
the 96-well experimental plate (Greiner 655180) containing the appropriate
medium (100
m
l per well) using a multichannel pipette.
5.
The experimental plate is then incubated at the desired temperature and
aeration conditions in a Synergy II plate reader (BioTek Germany, Bad
Friedrichshall, DE).
6.
Injections can be performed to all or selected wells at the desired time intervals
using the injectors of the Synergy II plate reader.
7.
User-defined protocols will determine the time intervals at which optical density
(OD
600
) and reporter protein emission readings are to be recorded. We typically
take readings every 10 min for the duration of the growth experiment, although
intervals as short as 1 min have also been successful (
Botella
et al.
, 2010
).
8.
Growth (OD
600
) and expression data are stored, then exported to an Excel
spreadsheet file for subsequent analysis.
m
7.6
Data analysis
Growth of the individual cultures in the 96-well plate is monitored by measuring
optical density at 600 nm (OD
600
), while fluorescence monitors expression of the
GFP protein. Because the length of the light path in a 96-well plate is shorter than
the 1-cm path-length in a standard spectrophotometer cuvette, the path-length is cor-
rected by taking an OD
900
and OD
977
reading for each well at the beginning of the
experiment. The path length is corrected by multiplying each OD
600
reading (after
