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: Mastermix, lysate, nuclease free water and DNA template<br/>
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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: Luciferase Reaction Mix, Dilution Reagent, Luciferse DNA, Cell-free expression Mix<br/>
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<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 30-45 min + running time <br/>
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<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>Mastermix</h4>
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<h4>Luciferase Reaction Reagent</h4>
 
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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"> stock concentration </th><th class="col2">amount of stock for 2 ml</th>
 
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<td class="col0"> Creatine phosphate </td><td class="col1"> 80 mM </td><td class="col2"> 4 </td>
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<td class="col0"> D-Luciferin </td><td class="col1"> 100 mM </td><td class="col2"> 10µl </td>
 
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<td class="col0"> Potassium glutamate </td><td class="col1"> 200 mM </td><td class="col2"> 3.7 </td>
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<td class="col0"> DTT </td><td class="col1"> 1 M </td><td class="col2"> 200 µl </td>
 
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<td class="col0"> HEPES-KOH pH 7.5 </td><td class="col1"> 55 mM </td><td class="col2"> 2.75 </td>
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<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"> Folinic acid </td><td class="col1"> 35 µg/ml </td><td class="col2"> 1.75 </td>
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<td class="col0"> BSA </td><td class="col1"> - </td><td class="col2"> 4 mg </td>
 
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<td class="col0"> Amino acids </td><td class="col1"> 2mM </td><td class="col2"> 1.25 </td>
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<td class="col0"> CoA </td><td class="col1"> 30 mM </td><td class="col2"> 0.6 µl </td>
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<td class="col0"> <em>E. coli</em> tRNA </td><td class="col1"> 175 µg/ml </td><td class="col2"> 1.14 </td>
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<td class="col0">PEG4000 </td><td class="col1"> 2% </td><td class="col2"> 1 </td>
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<td class="col0">DTT </td><td class="col1"> 1,7 mM </td><td class="col2"> 0.85 </td>
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<td class="col0"> ATP </td><td class="col1"> 1,2 mM </td><td class="col2"> 0.6 </td>
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<td class="col0">MgOAc </td><td class="col1"> 11 mM </td><td class="col2"> 0.55 </td>
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<td class="col0"> CTP </td><td class="col1"> 0.8 mM </td><td class="col2"> 0.4 </td>
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<td class="col0"> GTP </td><td class="col1"> 0.8 mM </td><td class="col2"> 0.4 </td>
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<td class="col0"> UTP </td><td class="col1"> 0.8 mM </td><td class="col2"> 0.4 </td>
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<td class="col0"> cAMP </td><td class="col1"> 0.65 mM</td><td class="col2"> 0.325 </td>
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<td class="col0">Creatine phosphokinase </td><td class="col1"> 80 mM</td><td class="col2"> 0.175 </td>
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<h4>Final reaction</h4>
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<h4>Dilution Reagent</h4>
 
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<li class="level1"><div class="li"> incubate 2-4 hrs at 37 °C in autoclaved tubes or 96 well plate (low bind)</div>
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<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.
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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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Revision as of 13:55, 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 stock concentration amount in 50 µl reaction [µl]
Master mix - 17.68
Lysate - 22.5
DNA at least 217 ng/µl 3250 - 5000 ng
H2O (nuclease free) - up to 50 µl

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

  • add creatine phosphokinase last to mastermix
  • work quickly, sterile and as nuclease free as possible (on ice)
  • add DNA last to start reactions simultaneously
  • amount of lysate used can be varied (here 45%)
  • the used chemicals should have a high grade of purity
  • Certain counterions like Chlor and Sodium should be avoided
  • feed: adding 10 mM Mg(OAc) every 20 minutes can increase reaction output profoundly

Reaction is adapted from the European Molecular Biology Laboratory (EMBL), Heidelberg, Germany