Getting Started
You’ve got PURE Protein Mix and you want to make PURE. You’ll need Energy Mix, the thirty-one (31) small molecules (e.g., rNTPs, amino acids, buffering salts, etc.) and tRNAs (E. coli) needed to fuel transcription and translation.
Here, we describe the components of energy mix, how to make it, and how to store it properly.
Reagent | Concentration in Energy Mix (mM) | Concentration in Final Reaction (mM) |
HEPES | 125 | 50 |
Potassium glutamate | 250 | 100 |
Magnesium acetate | 29.5 | 11.8 |
rNTPs (each) | 5 | 2 |
tRNA [mg/mL] | 8.75 | 3.5 |
Amino Acids (each) | 0.75 | 0.325 |
Creatine phosphate | 50 | 20 |
TCEP | 2.5 | 1 |
Folinic acid | 0.05 | 0.02 |
Spermidine | 5 | 2 |
Prerequisite Protocols
Make amino acid mix (3.25 mM):
Assemble Amino Acid Mix
Materials and Equipment
Name | Product | Manufacturer | Part # | Price | Storage Conditions | Link |
Potassium Hydroxide | Potassium hydroxide - ACS reagent, ≥85%, pellets | Sigma-Aldrich | 221473-1KG | $99.90 | 4C to 30C | [link] |
HEPES | HEPES, Crystalline Powder, ≥99.5% (titration), Poly bottle | Sigma-Aldrich | H3375-500G | $431 | 4C to 30C | [link] |
Potassium Glutamate | L-Glutamic acid potassium monohydrate | Sigma-Aldrich | G1501-100G | $74.80 | 4C to 30C | [link] |
Magnesium acetate | Magnesium acetate tetrahydrate | Sigma-Aldrich | M0631-100G | $34.80 | 4C to 30C | [link] |
rATP | Adenosine 5′-triphosphate disodium salt hydrate, BioXtra, ≥99% (HPLC), from microbial | Sigma-Aldrich | A7699-1G | $125.00 | -25C to -15C | [link] |
rGTP | Guanosine 5′-triphosphate sodium salt hydrate, ≥ 95% (HPLC), powder | Sigma-Aldrich | G8877-100MG | $155 | -25C to -15C | [link] |
rCTP | Cytidine 5′-triphosphate disodium salt, ≥95% | Sigma-Aldrich | C1506-100MG | $102.00 | -25C to -15C | [link] |
rUTP | Uridine 5′-triphosphate trisodium salt hydrate, Type IV, ≥93.0% (HPLC) | Sigma-Aldrich | U6750-100MG | $58.20 | -25C to -15C | [link] |
Creatine phosphate | Sodium creatine phosphate dibasic tetrahydrate, ≥98.0%(NT) | Sigma-Aldrich | 27920-1G | $102 | -25C to -15C | [link] |
TCEP-HCl (0.5M; presuspended) | Thermo Scientific, Bond-Breaker® Tris[2-Carboxyethyl]phosphine Neutral Solution (TCEP), Application=Water-Soluble Reducing Agent, Molecular Weight=286.65, Size=5 mL | Thermo Scientific | 77720 | $175.65 | [link] | 4C to 30C |
Folinic acid | Folinic acid calcium salt hydrate | Sigma-Aldrich | F7878-100MG | $108 | 4C to 30C | [link] |
Spermidine | Spermidine, ≥99% (GC) | Sigma-Aldrich | S2626-5G | $176 | -25C to -15C | [link] |
tRNAs | Ribonucleic acid, transfer from Escherichia coli (Type XX, Strain W, lyophilized) | Sigma-Aldrich | R1753-100UN | $69.00 | -25C to -15C | [link] |
Amino Acids | Amino Acid Mix (3.25 mM; see | -80C to -20C |
Protocol
Make the following stock solutions
Component | MW [g/mol] | Amount [g] | Final Volume [mL] | Storage | Needs pH adjustment? | Needs Sterilization? |
Potassium Hydroxide (1M) | 56.11 | 14.0 | 250 | 4C to 30C | no | no |
HEPES-KOH (pH=7.6; 1M) | 238.3 | 59.5 | 250 | 4C to 30C; dark | yes | no |
Potassium glutamate (2.5M) | 203.23 | 21.8 | 50 | 4C to 30C | no | no |
Magnesium acetate (1M) | 214.45 | 10.8 | 50 | 4C to 30C | no | no |
Creatine phosphate (500 mM) | 327.14 | 1 | 9.43 | -25C to -15C | no | no |
Folinic acid (5 mM) | 511.50 | 0.119 | 50 | -25C to -15C | no | no |
Spermidine (200 mM) | 145.25 | 3.64 | 50 | -25C to -15C | no | no |
Amino acid mix | -85C to -15C | yes | yes |
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Assemble components of Energy Mix (except tRNAs)
Component | Stock Concentration [mM] | Concentration in Energy Mix [mM] | Volume to Add [uL] |
HEPES | 1000 | 125 | 62.5 |
Potassium glutamate | 2500 | 250 | 50.0 |
Magnesium acetate | 1000 | 29.5 | 14.8 |
rNTPs (each) | 25 | 5 | 100.0 |
Creatine phosphate | 500 | 50 | 50.0 |
TCEP | 500 | 2.5 | 2.5 |
Folinic acid | 5 | 0.05 | 5.0 |
Spermidine | 500 | 5 | 5.0 |
Amino Acid solution | 3.25 | 0.75 | 115.4 |
Water | n/a | n/a | 94.9 |
Total | 500 |
Open a fresh vial of tRNAs (100 U); pipette 476 uL of Energy Mix (except tRNAs) into the vial. Mix by pipetting briefly until tRNAs are resuspended.
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Aliquot Energy Mix into 1.5 mL microfuge tubes (between 50 uL and 100 uL per aliquot).
Store Energy Mix aliquots frozen (-75C and -85C).
Resources and References
The OnePot PURE methods paper (JOVE) has great interactive tables!
- Use Supplementary Table 4 for making stock solutions
- Use Supplementary Table 5 for assembling Energy Mix
OnePot-JOVE-Table-S4-stock-solutions.xlsx11.5KB
OnePot-JOVE-Table-S5-energy-mix-worksheet.xlsx10.9KB
- Papers
- Original PURE paper
- OnePot PURE
- Resources
- NEB absorbance units calculator
Credits
- Developers