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

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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"> Amino acids </td><td class="col1"> 2 mM </td><td class="col2"> 1.25 </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">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"> ATP </td><td class="col1"> 1.2 mM </td><td class="col2"> 0.6 </td>
 
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Revision as of 18:42, 16 September 2015

""

Protocol Cell-free expression

How to express proteins cell-free from DNA templates

material: Mastermix, lysate, nuclease free water and DNA template
duration: Preparation 30-45 min + running time

Mastermix

Component stock concentration amount in 50 µl reaction [µl]
Creatine phosphate 80 mM 4
Potassium glutamate 200 mM 3.7
HEPES-KOH pH 7.5 55 mM 2.75
Folinic acid 35 µg/ml 1.75
Amino acids 2 mM 1.25
E. coli tRNA 175 µg/ml 1.14
PEG4000 2% 1
DTT 1.7 mM 0.85
ATP 1.2 mM 0.6
MgOAc 11 mM 0.55
CTP 0.8 mM 0.4
GTP 0.8 mM 0.4
UTP 0.8 mM 0.4
cAMP 0.65 mM 0.325
Creatine phosphokinase 80 mM 0.175

Final reaction

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

  • incubate 2-4 hrs at 37 °C in autoclaved tubes or 96 well plate (low bind)

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