Difference between revisions of "Team:Freiburg/Protocols/LUC"
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− | <a class="media" href="https://static.igem.org/mediawiki/2015/4/4b/Freiburg_wiki-symbols-orga.png" title="wiki:symbols:orga.png"><img alt="" class="media" src="https://static.igem.org/mediawiki/2015/4/4b/Freiburg_wiki-symbols-orga.png"/></a> material: | + | <a class="media" href="https://static.igem.org/mediawiki/2015/4/4b/Freiburg_wiki-symbols-orga.png" title="wiki:symbols:orga.png"><img alt="" class="media" src="https://static.igem.org/mediawiki/2015/4/4b/Freiburg_wiki-symbols-orga.png"/></a> material: Luciferase Reaction Mix, Dilution Reagent, Luciferse DNA, Cell-free expression Mix<br/> |
− | <a class="media" href="https://static.igem.org/mediawiki/2015/b/bb/Freiburg_wiki-symbols-alarm2.png" title="wiki:symbols:alarm2.png"><img alt="" class="media" src="https://static.igem.org/mediawiki/2015/b/bb/Freiburg_wiki-symbols-alarm2.png"/></a> duration: Preparation | + | <a class="media" href="https://static.igem.org/mediawiki/2015/b/bb/Freiburg_wiki-symbols-alarm2.png" title="wiki:symbols:alarm2.png"><img alt="" class="media" src="https://static.igem.org/mediawiki/2015/b/bb/Freiburg_wiki-symbols-alarm2.png"/></a> duration: Preparation 20 min + running time <br/> |
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− | <h4> | + | <h4>Luciferase Reaction Reagent</h4> |
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− | <th class="col0">Component </th><th class="col1"> stock concentration </th><th class="col2">amount | + | <th class="col0">Component </th><th class="col1"> stock concentration </th><th class="col2">amount of stock for 2 ml</th> |
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<tr class="row1"> | <tr class="row1"> | ||
− | <td class="col0"> | + | <td class="col0"> D-Luciferin </td><td class="col1"> 100 mM </td><td class="col2"> 10 µl </td> |
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<tr class="row2"> | <tr class="row2"> | ||
− | <td class="col0"> | + | <td class="col0"> DTT </td><td class="col1"> 1 M </td><td class="col2"> 200 µl </td> |
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− | <td class="col0"> | + | <td class="col0"> ATP </td><td class="col1"> 100 mM </td><td class="col2"> 12.5 µl </td> |
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− | <td class="col0"> | + | <td class="col0"> BSA </td><td class="col1"> - </td><td class="col2"> 4 mg </td> |
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<tr class="row5"> | <tr class="row5"> | ||
− | <td class="col0"> | + | <td class="col0"> CoA </td><td class="col1"> 30 mM </td><td class="col2"> 0.6 µl </td> |
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− | <th class="col0">Component </th><th class="col1"> | + | <th class="col0">Component </th><th class="col1"> end concentration </th> |
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− | <td class="col0"> | + | <td class="col0">TRIS-phosphate, pH 7.8 </td><td class="col1"> 25 M</td> |
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<tr class="row2"> | <tr class="row2"> | ||
− | <td class="col0"> | + | <td class="col0"> DTT </td><td class="col1"> 2 mM </td> |
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− | <td class="col0"> | + | <td class="col0"> EDTA </td><td class="col1"> 2 mM </td> |
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− | <td class="col0"> | + | <td class="col0"> Glycerol </td><td class="col1"> 10% </td> |
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+ | <td class="col0"> TritonX100 </td><td class="col1"> 1% </td> | ||
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+ | <td class="col0"> BSA </td><td class="col1"> 1 mg/ml </td> | ||
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+ | <li class="level1"><div class="li"> Both solutions can be stored at -20°C </div> | ||
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− | <li class="level1"><div class="li"> | + | <li class="level1"><div class="li"> To characterize a reaction, the luciferase plasmid was added to the expression mix and the expression is run using desired conditions (standard: 2 h at 37°C, no shaking). Afterwards, the reaction is stopped by putting it on ice. Inside a well of a 96-well microplate, 20 µl of the reaction are mixed with 20 µl of dilution reagent, creating a 1:2 dilution. If a 1:4 dilution is demanded, 20 µl of the first dilution are added to another 20 µl dilution reagent, otherwise they are discard-ed. 50 µl of the luciferase assay reagent is added to the reaction only seconds before the measurement is started. |
+ | The microplate reader is set to perform a full spectrum emission scan (400 - 700 nm) of each well. Because of the kinetic temperature dependency of luciferase, it was minded to perform every scan at 25°C. | ||
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− | <li class="level1"><div class="li"> | + | <li class="level1"><div class="li"> The Reaction Reagent tends to get contaminated so regular preparation is necessary</div> |
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Latest revision as of 07:11, 20 November 2015
Protocol Cell-free expression
How to perform a Luciferase assay
material: Luciferase Reaction Mix, Dilution Reagent, Luciferse DNA, Cell-free expression Mix
duration: Preparation 20 min + running time
Luciferase Reaction Reagent
Component | stock concentration | amount of stock for 2 ml |
---|---|---|
D-Luciferin | 100 mM | 10 µl |
DTT | 1 M | 200 µl |
ATP | 100 mM | 12.5 µl |
BSA | - | 4 mg |
CoA | 30 mM | 0.6 µl |
Dilution Reagent
Component | end concentration |
---|---|
TRIS-phosphate, pH 7.8 | 25 M |
DTT | 2 mM |
EDTA | 2 mM |
Glycerol | 10% |
TritonX100 | 1% |
BSA | 1 mg/ml |
- Both solutions can be stored at -20°C
Implementation
- To characterize a reaction, the luciferase plasmid was added to the expression mix and the expression is run using desired conditions (standard: 2 h at 37°C, no shaking). Afterwards, the reaction is stopped by putting it on ice. Inside a well of a 96-well microplate, 20 µl of the reaction are mixed with 20 µl of dilution reagent, creating a 1:2 dilution. If a 1:4 dilution is demanded, 20 µl of the first dilution are added to another 20 µl dilution reagent, otherwise they are discard-ed. 50 µl of the luciferase assay reagent is added to the reaction only seconds before the measurement is started. The microplate reader is set to perform a full spectrum emission scan (400 - 700 nm) of each well. Because of the kinetic temperature dependency of luciferase, it was minded to perform every scan at 25°C.
Remarks
- The Reaction Reagent tends to get contaminated so regular preparation is necessary