Team:Freiburg/Protocols/prepLysate
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Protocol Cell-free expression
Preparation of a +Lysate for Cell-free exxpression
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 | 2mM | 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