Team:BIOTEC Dresden/ProtocolsAHL assay

From 2010.igem.org

(Difference between revisions)
(Removing all content from page)
 
(4 intermediate revisions not shown)
Line 1: Line 1:
-
{{Biotec_Dresden/Header}}
 
-
<html>
 
-
<body>
 
-
Preparing a 96-well plate for HHL assay
 
-
 
-
1. Set-up
 
-
 
-
Column1: 180µl LB-medium + 20µl H2O
 
-
Column2: 180µl cells + 20µl H2O
 
-
Column3: 200µl cells in 0.01nM HHL
 
-
Column4: 200µl cells in 0.1nM HHL
 
-
Column5: 200µl cells in 1nM HHL
 
-
Column6: 200µl cells in 10nM HHL
 
-
Column7: 200µl cells in 50nM HHL
 
-
Column8: 200µl cells in 100nM HHL
 
-
Column9: 200µl cells in 200nM HHL
 
-
Column10: 200µl cells in 500nM HHL
 
-
Column11: 200µl cells in 1000nM HHL
 
-
Column12: 200µl cells in 2000nM HHL
 
-
 
-
2. Pipetting scheme
 
-
 
-
2.1. Stock solutions
 
-
 
-
Final con. / µM Stock used / µM Vol. of stock / µl Vol. of water / µl
 
-
150 15000 12 1188
 
-
15 150 120 1080
 
-
1.5 15 120 1080
 
-
0.15 1.5 120 1080
 
-
0.015 0.15 120 1080
 
-
 
-
2.2. 94-well plate
 
-
 
-
The volumes below are sufficient for pipetting four well plates at a time.
 
-
 
-
Final con. / nM Stock used / µM Vol. of stock / µl Vol. of water / µl
 
-
0.01 0.015 4.8 715.2
 
-
0.1 0.015 48 672
 
-
1 0.15 48 672
 
-
10 0.15 480 240
 
-
50 1.5 240 480
 
-
100 1.5 480 240
 
-
200 15 96 624
 
-
500 15 240 480
 
-
1000 15 480 240
 
-
2000 150 96 624
 
-
 
-
Pipet 20µl of the above concentrations into the corresponding wells and add 180µl of cell suspension right before the measurement of fluorescence.
 
-
 
-
 
-
3. Tecan plate reader
 
-
 
-
Start the machine 20 minutes in advance and heat the chamber to 37°C. Put in the plate and start the measurement with the following setting:
 
-
 
-
• Measure OD at 612 nm
 
-
• Measure fluorescence with an excitation wavelength of 485nm and an emission wavelength of 485nm for both GFP and YFP
 
-
• Shake
 
-
• Repeat measurements every 5min for 3 hours
 
-
 
-
4. Data processing
 
-
 
-
• Normalize the data by dividing all fluorescence data by the corresponding optical density
 
-
• Subtract the obtained value of the reference column (column 2) from the calculated relative fluorescence of the wells containing HHL
 
-
• Plot the fluorescence data over the time
 
-
 
-
 
-
 
-
</body>
 
-
</html>
 
-
 
-
[[Category:BIOTEC Dresden/Protocol|AHL assay]]
 
-
{{Biotec_Dresden/Bottom}}
 

Latest revision as of 02:39, 28 October 2010