Team:BIOTEC Dresden/ProtocolsAHL assay

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(New page: {{Biotec_Dresden/Header}} <html> <body> <div id="content_prim"> <div id="materials"> <h2>Materials</h2> <ul> <li>Escherichia coli DH5 alpha chemical competent cells</li> <li>1mL SOC (room ...)
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<h2>Materials</h2>
 
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<ul>
 
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<li>Escherichia coli DH5 alpha chemical competent cells</li>
 
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<li>1mL SOC (room temperature) for each reaction</li>
 
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<li>Ice</li>
 
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<li>Plasmid DNA</li>
 
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<li>Heating block</li>
 
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<li>1.5ml microfuge tube</li>
 
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<li>LB-agar plate with corresponding antibiotic</li>
 
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<div class="visualClear"></div>
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Preparing a 96-well plate for HHL assay
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<div id="procedure">
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<h2>Procedure</h2>
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1. Set-up
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<ul>
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<li>Chill DNA samples and tubes on ice.
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Column1: 180µl LB-medium + 20µl H2O
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<li>Place LB-agar plates in <span class="markup temp">37°C</span> incubator to warm.
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Column2: 180µl cells + 20µl H2O
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<li>Remove chemical competent cells from <span class="markup temp">-80°C</span> freezer and thaw on ice. Alternatively, freshly prepared chemical competent cells may be used immediately.</li>
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Column3: 200µl cells in 0.01nM HHL
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<li>Dial a P2 pipetman to either <span class="markup volume">1 or 2μL</span> depending on the salt content of your DNA sample. Use <span class="markup volume">2μL</span> for samples that have been purified in some way.</li>
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Column4: 200µl cells in 0.1nM HHL
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<li>Dial a P200 pipetman to <span class="markup volume">50μL</span> or whatever volume of chemical competent cells you want to use; usually <span class="markup volume">20-50μL</span> .</li>
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Column5: 200µl cells in 1nM HHL
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<li>Pipet <span class="markup volume">1-2μL</span> of DNA sample and add to chemical competent cells. Swirl tip around gently in cells to mix DNA and cells. Do not pipet up and down.</li>
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Column6: 200µl cells in 10nM HHL
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<li>Place the mix back on <span class="markup temp">ice</span> for <span class="markup time">30 mins</span>.</li>
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Column7: 200µl cells in 50nM HHL
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<li>Pulse the cells with a heat shock by placing the microfuge tubes in a heating block for <span class="markup time">45 seconds</span> at <span class="markup temp">42°C</span>.</li>
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Column8: 200µl cells in 100nM HHL
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<li>Place sample on ice for <span class="markup time">2 mins</span> and then add SOC medium. This step should be done as quickly as possible to prevent cells from dying off.</li>
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Column9: 200µl cells in 200nM HHL
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<li>Chill sample on ice for <span class="markup time">2 mins</span> to permit the cells to recover.</li>
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Column10: 200µl cells in 500nM HHL
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<li>Transfer eppendorf tube to <span class="markup temp">37°C</span> incubator and shake to promote aeration. Incubate for <span class="markup time">1 hr</span> to permit expression of antibiotic resistance gene.</li>
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Column11: 200µl cells in 1000nM HHL
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<li>Plate transformation onto prewarmed LB-agar plate supplemented with appropriate antibiotic.</li>
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Column12: 200µl cells in 2000nM HHL
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2. Pipetting scheme
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<h2>Reference</h2>
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<p>adapted from <a href="http://openwetware.org/wiki/Knight:Electroporation">http://openwetware.org/wiki/Knight:Electroporation</a></p>
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2.1. Stock solutions
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</div>
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Final con. / µM Stock used / µM Vol. of stock / µl Vol. of water / µl
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150 15000 12 1188
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15 150 120 1080
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1.5 15 120 1080
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0.15 1.5 120 1080
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0.015 0.15 120 1080
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2.2. 94-well plate
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The volumes below are sufficient for pipetting four well plates at a time.
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Final con. / nM Stock used / µM Vol. of stock / µl Vol. of water / µl
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0.01 0.015 4.8 715.2
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0.1 0.015 48 672
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1 0.15 48 672
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10 0.15 480 240
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50 1.5 240 480
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100 1.5 480 240
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200 15 96 624
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500 15 240 480
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1000 15 480 240
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2000 150 96 624
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Pipet 20µl of the above concentrations into the corresponding wells and add 180µl of cell suspension right before the measurement of fluorescence.
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3. Tecan plate reader
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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:
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• Measure OD at 612 nm
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• Measure fluorescence with an excitation wavelength of 485nm and an emission wavelength of 485nm for both GFP and YFP
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• Shake
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• Repeat measurements every 5min for 3 hours
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4. Data processing
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• Normalize the data by dividing all fluorescence data by the corresponding optical density
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• Subtract the obtained value of the reference column (column 2) from the calculated relative fluorescence of the wells containing HHL
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• Plot the fluorescence data over the time
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Revision as of 00:52, 28 October 2010

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
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