Oct 17, 2019
Energy solution preparation for OnePot PURE cell-free system
- 1EPFL - EPF Lausanne
- iGEM EPFL
External link: https://pubs.acs.org/doi/10.1021/acssynbio.8b00427
Protocol Citation: Konstantinos Ragios 2019. Energy solution preparation for OnePot PURE cell-free system. protocols.io https://dx.doi.org/10.17504/protocols.io.8ayhsfw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 16, 2019
Last Modified: October 17, 2019
Protocol Integer ID: 28728
Keywords: energy, PURE, cell-free, iGEM, expression, tRNA, NTPs
Abstract
In this protocol we explain the procedure to create the Energy solution used for protein expression in OnePot PURE cell-free system.
Materials
Material/Consumables:
- Amino Acids (Sigma-Aldrich: LAA21-1KT)
- Magnesium acetate (Sigma-Aldrich: M0631)
- Potassium glutamate (Sigma-Aldrich: 49601)
- DTT (SantaCruz Biotech: sc-29089B)
- ATP (ThermoFisher: R0481)
- GTP (ThermoFisher: R0481)
- CTP (ThermoFisher: R0481)
- UTP (ThermoFisher: R0481)
- tRNA (Roche:10109541001)
- Creatine phosphate (Sigma-Aldrich: 27920)
- Folinic acid (Sigma-Aldrich: PHR1541)
- Spermidine (Sigma-Aldrich: S2626)
- HEPES (Sigma-Aldrich: H0887-100ML)
- biDistilled water
Equipment:
- Vortex
- Nanodrop Spectrophotometer
Before start
Keep all the components in ice while preparing the Energy solution.
For a 2.5x Energy Solution add the materials needed tho a tube. The final concentration of the components is presented in Table 1.
Note
Before adding each component make sure it is totally melted and then vortex for a few seconds
| Compound | Concentration | Units | |
| Amino Acids | 0.75 | mM | |
| Magnesium acetate | 29.5 | mM | |
| Potassium glutamate | 250 | mM | |
| DTT | 2.5 | mM | |
| ATP | 5 | mM | |
| GTP | 5 | mM | |
| CTP | 2.5 | mM | |
| UTP | 2.5 | mM | |
| tRNA | 130 | UA260/mL | |
| Creatine phosphate | 50 | mM | |
| Folinic acid | 0.05 | mM | |
| Spermidine | 5 | mM | |
| HEPES | 125 | mM |
Note
Because tRNA degrades over time before adding it to the solution you need to check the A260 units.
To complete the solution add biDistilled water until you reach the desirable volume, and then vortex for a few seconds
The Energy solution is stored at -80o C.