Difference between revisions of "Team:Freiburg/Protocols/LUC"

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<th class="col0">Component </th><th class="col1"> stock concentration </th><th class="col2">amount in 50 µl reaction [µl]</th>
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<th class="col0">Component </th><th class="col1"> end concentration </th>
 
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<td class="col0"> Master mix </td><td class="col1"> - </td><td class="col2"> 17.68 </td>
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<td class="col0">TRIS-phosphate, pH 7.8 </td><td class="col1"> 25M</td>
 
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<td class="col0"> Lysate </td><td class="col1"> - </td><td class="col2"> 22.5 </td>
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<td class="col0"> DTT </td><td class="col1"> 2mM </td>
 
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<tr class="row3">
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<td class="col0"> DNA </td><td class="col1"> at least 217 ng/µl </td><td class="col2"> 3250 - 5000 ng </td>
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<td class="col0"> EDTA </td><td class="col1"> 2mM </td>
 
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</tr>
 
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<td class="col0"> H2O (nuclease free) </td><td class="col1"> - </td><td class="col2"> up to 50 µl </td>
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<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"> 1mg/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>
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<li class="level1"><div class="li"> add creatine phosphokinase last to mastermix</div>
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<li class="level1"><div class="li"> The Reaction Reagent tends to get contaminated so regular preparation is necessary</div>
 
</li>
 
</li>
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<li class="level1"><div class="li"> work quickly, sterile and as nuclease free as possible (on ice)</div>
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</li>
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<li class="level1"><div class="li"> add DNA last to start reactions simultaneously</div>
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</li>
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<li class="level1"><div class="li"> amount of lysate used can be varied (here 45%)</div>
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</li>
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<li class="level1"><div class="li"> the used chemicals should have a high grade of purity</div>
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<li class="level1"><div class="li"> Certain counterions like Chlor and Sodium should be avoided</div>
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</li>
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<li class="level1"><div class="li"> feed: adding 10 mM Mg(OAc) every 20 minutes can increase reaction output profoundly</div>
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<p>
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<strong>Reaction is adapted from the European Molecular Biology Laboratory (EMBL), Heidelberg, Germany </strong>
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<div class="tags"><span>
 
<div class="tags"><span>
 
<a class="wikilink1" href="/igem2015/doku.php?id=tag:info&amp;do=showtag&amp;tag=info" rel="tag" title="tag:info">info</a>
 
<a class="wikilink1" href="/igem2015/doku.php?id=tag:info&amp;do=showtag&amp;tag=info" rel="tag" title="tag:info">info</a>

Revision as of 21:37, 14 September 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 10µl
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 25M
DTT 2mM
EDTA 2mM
Glycerol 10%
TritonX100 1%
BSA 1mg/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