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| Line 1: |
Line 1: |
| '''Aims'''
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| *To test to see if the DNA constructs from the registry are viable. This is done in vivo.
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| *The constructs are pTet-GFP, pT7-GFP and pcI-GFP.
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| ===Day 1===
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| ====Equipment====
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| *7ml sterile tubes x4
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| *1.5ml Eppendorf tube x1
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| *Room temperature 25<sup>o</sup>C
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| *Gilson Pipettes
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|
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| ====Reagents====
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| *''E.coli'' BL21; culture containing parts :pTet-GFP, pT7-GFP, pcI-GFP
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| *LB medium
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|
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| *Ampicillin stock (50 mg/ml)
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| *GFP Standard Solution - This concentration was unknown, however because we only wanted it as a positive control to show there was fluorescence and that the flurometer could measure it
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| <br>
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|
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| ====Protocol====
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| '''Innoculation of Media'''
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| #Inoculate 10ul of transformed E.coli cells in individual 2ml LB medium containing 2ul of ampicillin
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| #Incubate at 37°C for overnight in a shaker. '''(This is to get a good stock of cells for use in the experiment. After the overnight culture the cells will be in stationary phase)'''
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| <br>
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|
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| ===Day 2===
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| ====Equipment====
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| *Well-plate x1
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| *Fluorometer
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| *Gilson pipettes 1000 and 200
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| *Eppendorf tubes
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|
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| ====Reagents====
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| *ddH2O
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| *GFP standard solution
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| <br>
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|
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| ====Protocol====
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| '''Preparation of diluted GFP standard solution'''<br>
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| #Add 995ul of ultra pure water an eppendorf tube, together with 5ul of undiluted GFP standard solution and mix. '''(This gives a 200x dilution to be used as a positive control)'''
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| #Place the tube on ice till it is ready to be used.
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| <br>
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| <br>
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|
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|
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| '''Loading Plate'''
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| #Transfer 200 µl aliquots of each of the cultures to a flat-bottomed 96 well plate. '''(Follow the schematic as shown)'''
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| #Three wells to be filled with 200µl of media to measure the absorbance background.
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| #Standard GFP solution added as a positive control.
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| #Remove lid and measure in the flourometer.
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| : (Fluorescence measurements - 488 nm excitation filter, 525 nm emission filter, 0.5 seconds, CW lamp energy 12901 units)
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| #Repeat the measurement a further two times straight after each other '''(This is to test the variability of the machine)'''
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| <br>
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| <br>
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| <br>
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|
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| '''Schematic'''
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|
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|
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| {| border="1" cellpadding="1"
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| {| border="1" cellpadding="2"
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| !<u>Well </u> || <u>Test Construct</u> || <u>Stock Volume (ul)</u> !! <u>AHL (ul)</u> !! <u>Final [AHL]</u>
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| |-
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| |<font color="#8080ff">A1
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| |<font color="#8080ff">pTet-GFP
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| |<font color="#8080ff">200
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| |0
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| |0
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| |-
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| |<font color="#8080ff">A2
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| |<font color="#8080ff">pTet-GFP
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| |<font color="#8080ff">200
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| |0
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| |0
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| |-
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| |<font color="#8080ff">A3
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| |<font color="#8080ff">pTet-GFP
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| |<font color="#8080ff">200
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| |0
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| |0
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| |-
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| |<font color="#008000">A5
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| |<font color="#008000">LB-Amp Media
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| |<font color="#008000">200
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| |0
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| |0
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| |-
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| |<font color="#008000">A6
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| |<font color="#008000">LB-Amp Media
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| |<font color="#008000">200
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| |0
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| |0
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| |-
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| |<font color="#008000">A7
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| |<font color="#008000">LB-Amp Media
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| |<font color="#008000">200
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| |0
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| |0
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| |-
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| |<font color="#800080">B1
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| |<font color="#800080">pT7-GFP
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| |<font color="#800080">200
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| |0
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| |0
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| |-
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| |<font color="#800080">B2
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| |<font color="#800080">pT7-GFP
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| |<font color="#800080">200
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| |0
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| |0
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| |-
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| |<font color="#800080">B3
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| |<font color="#800080">pT7-GFP
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| |<font color="#800080">200
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| |0
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| |0
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| |-
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| |<font color="#455f3d">B5
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| |<font color="#455f3d">LB-Amp Media + Non-expressing culture
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| |<font color="#455f3d">200
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| |0
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| |0
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| |-
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| |<font color="#455f3d">B6
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| |<font color="#455f3d">LB-Amp Media + Non-expressing culture
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| |<font color="#455f3d">200
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| |0
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| |0
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| |-
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| |<font color="#455f3d">B7
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| |<font color="#455f3d">LB-Amp Media + Non-expressing culture
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| |<font color="#455f3d">200
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| |0
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| |0
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| |-
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| |<font color="#d7004d"> C1
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| |<font color="#d7004d"> pcI-GFP
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| |<font color="#d7004d"> 200
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| |0
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| |0
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| |-
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| |<font color="#d7004d"> C2
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| |<font color="#d7004d"> pcI-GFP
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| |<font color="#d7004d"> 200
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| |0
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| |0
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| |-
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| |<font color="#d7004d">C3
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| |<font color="#d7004d">pcI-GFP
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| |<font color="#d7004d"> 200
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| |0
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| |0
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| |-
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| |<font color="#80f05b">C5
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| |<font color="#80f05b">Diluted GFP Solution
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| |<font color="#80f05b">200
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| |0
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| |0
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| |-
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| |<font color="#80f05b">C6
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| |<font color="#80f05b">Diluted GFP Solution
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| |<font color="#80f05b">200
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| |0
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| |0
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| |-
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| |<font color="#80f05b">C7
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| |<font color="#80f05b">Diluted GFP Solution
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| |<font color="#80f05b">200
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| |2
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| |0
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| |-
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| |<font color="#00afad"> D1
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| |<font color="#00afad"> pTet-LuxR-pLux-GFP + 10μM AHL
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| |<font color="#00afad"> 200
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| |2
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| |10<sup>-7</sup>M
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| |-
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| |<font color="#00afad"> D2
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| |<font color="#00afad"> pTet-LuxR-pLux-GFP + 10μM AHL
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| |<font color="#00afad"> 200
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| |2
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| |10<sup>-7</sup>M
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| |-
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| |<font color="#00afad"> D3
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| |<font color="#00afad"> pTet-LuxR-pLux-GFP + 10μM AHL
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| |<font color="#00afad"> 200
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| |2
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| |10<sup>-7</sup>M
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| |-
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| |<font color="#00625a">E1
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| |<font color="#00625a"> pTet-LuxR-pLux-GFP + 100μM AHL
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| |<font color="#00625a"> 200
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| |2
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| |10<sup>-6</sup>M
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| |-
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| |<font color="#00625a">E2
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| |<font color="#00625a">pTet-LuxR-pLux-GFP + 100μM AHL
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| |<font color="#00625a">200
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| |2
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| |10<sup>-6</sup>M
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| |-
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| |<font color="#00625a">E3
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| |<font color="#00625a">pTet-LuxR-pLux-GFP + 100μM AHL
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| |<font color="#00625a">200
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| |2
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| |10<sup>-6</sup>M
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| |}
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| [[Image:Icgems_invivo-testing_thur.png|450px|top|In vivo Testing 96 well plate]]
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| |}
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| <br>
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