Overview¶
Small Molecule Mix (SMix) is a combination of thirty-one (31) small molecules (e.g., rNTPs, amino acids, buffering salts, etc.) needed to fuel transcription and translation. Here, we describe the components of SMix, how to make it, and how to store it properly.
Important Information
Please read this section carefully. It contains important notes, resources, and safety information. Not all information included here is included in the lab-ready protocol.
Prerequisite Documentation
Composition
| Reagent | Concentration in Small Molecule Mix (mM) | Concentration in Final Reaction (mM) |
|---|---|---|
| HEPES-KOH (pH 7.6) | 166.67 | 50 |
| Potassium glutamate | 333.33 | 100 |
| Magnesium acetate | 25 | 7.5 |
| rATP | 6.67 | 2 |
| rGTP | 6.67 | 2 |
| rCTP | 3.33 | 1 |
| rUTP | 3.33 | 1 |
| Amino Acids (each) | 1.00 | 0.3 |
| Creatine phosphate | 66.67 | 20 |
| Folinic acid | 0.067 | 0.02 |
| Spermidine | 6.67 | 2 |
| TCEP | 3.33 | 1 |
Materials and Equipment¶
Bill of Materials
| Name | Category | Product | Manufacturer | Part # | Price | Storage | Link |
|---|---|---|---|---|---|---|---|
| Potassium Hydroxide | Salts | Potassium hydroxide - ACS reagent, ≥85%, pellets | Sigma-Aldrich | 221473-1KG | $99.90 | 4 °C to 30 °C | link |
| Hydrochloric acid (1 M) | Salts | Hydrochloric acid 1 N, Reagent Grade, 500 mL | VWR | E447-500L | $72.46 | 4 °C to 30 °C | link |
| HEPES | Salts | HEPES, Crystalline Powder, ≥99.5% (titration), Poly bottle | Sigma-Aldrich | H3375-500G | $431 | 4 °C to 30 °C | link |
| Potassium Glutamate | Salts | L-Glutamic acid potassium monohydrate | Sigma-Aldrich | G1501-100G | $74.80 | 4 °C to 30 °C | link |
| Magnesium acetate | Salts | Magnesium acetate tetrahydrate | Sigma-Aldrich | M0631-100G | $34.80 | 4 °C to 30 °C | link |
| rATP | NTP | Adenosine 5′-triphosphate disodium salt hydrate, BioXtra, ≥99% (HPLC) | Sigma-Aldrich | A7699-1G | $125.00 | -25 °C to -15 °C | link |
| rGTP | NTP | Guanosine 5′-triphosphate sodium salt hydrate, ≥95% (HPLC), powder | Sigma-Aldrich | G8877-100MG | $155 | -25 °C to -15 °C | link |
| rCTP | NTP | Cytidine 5′-triphosphate disodium salt, ≥95% | Sigma-Aldrich | C1506-100MG | $102.00 | -25 °C to -15 °C | link |
| rUTP | NTP | Uridine 5′-triphosphate trisodium salt hydrate, Type IV, ≥93.0% (HPLC) | Sigma-Aldrich | U6750-100MG | $58.20 | -25 °C to -15 °C | link |
| Creatine phosphate | Reagent | Sodium creatine phosphate dibasic tetrahydrate, ≥98.0%(NT) | Sigma-Aldrich | 27920-1G | $102 | -25 °C to -15 °C | link |
| TCEP-HCl (500 mM) | Reagent | Bond-Breaker® TCEP Neutral Solution, MW 286.65, 5 mL | Thermo Scientific | 77720 | $175.65 | 4 °C to 30 °C | link |
| Folinic acid | Reagent | Folinic acid calcium salt hydrate | Sigma-Aldrich | F7878-100MG | $108 | 4 °C to 30 °C | link |
| BME | Reagent | 2-Mercaptoethanol | Sigma-Aldrich | M6250-100ML | $65.50 | 4 °C to 30 °C | link |
| Spermidine | Reagent | Spermidine, ≥99% (GC) | Sigma-Aldrich | S2626-5G | $176 | -25 °C to -15 °C | link |
| Amino Acids | Biologic | Amino Acid Mix (3.25 mM) | b.next | — | — | -80 °C to -20 °C | Make Amino Acid Mix |
Protocol¶
Prepare Folinic Acid Stock (5 mM).¶
Weigh 12.5 mg folinic acid.
Dissolve to a final volume of 4.89 mL.
Aliquot and freeze at -20 °C.
Make All Other Stock Solutions.¶
Use the table below to prepare the specified stock solutions:
| Reagent | MW (g/mol) | Amount (g) | Final Volume (mL) | Storage | Needs pH adjustment? | Needs Sterilization? |
|---|---|---|---|---|---|---|
| Potassium Hydroxide (1 M) | 56.11 | 14.0 | 250 | 4 °C to 30 °C | no | no |
| HEPES-KOH (pH 7.6, 1 M) | 238.3 | 59.5 | 250 | 4 °C to 30 °C; dark | yes | no |
| Potassium glutamate (2.5 M) | 203.23 | 21.8 | 50 | 4 °C to 30 °C | no | no |
| Magnesium acetate (1 M) | 214.45 | 10.8 | 50 | 4 °C to 30 °C | no | no |
| Creatine phosphate (500 mM) | 327.14 | 1 | 9.43 | -25 °C to -15 °C | no | no |
| Folinic acid (5 mM) | 511.50 | See above | -25 °C to -15 °C | no | no | |
| Spermidine (500 mM) | 145.25 | 1 | 13.77 | -25 °C to -15 °C | no | no |
| Amino Acid Mix | see Make Amino Acid Mix | -85 °C to -15 °C | yes | yes |
Assemble Small Molecule Mix Components.¶
Use the table below to combine the previously prepared stock solutions into Small Molecule Mix:
| Reagent | Stock Concentration (mM) | Concentration in Small Molecule Mix (mM) | Volume to Add (uL) |
|---|---|---|---|
| HEPES-KOH (pH 7.6) | 1000 | 166.67 | 83.33 |
| Potassium glutamate | 2500 | 333.33 | 66.67 |
| Magnesium acetate | 1000 | 25 | 12.5 |
| rATP | 100 | 6.67 | 33.33 |
| rGTP | 100 | 6.67 | 33.33 |
| rCTP | 100 | 3.33 | 16.67 |
| rUTP | 100 | 3.33 | 16.67 |
| Creatine phosphate | 500 | 66.67 | 66.67 |
| TCEP | 500 | 3.33 | 3.33 |
| Folinic acid | 5 | 0.067 | 6.67 |
| Spermidine | 500 | 6.67 | 6.67 |
| Amino Acid Mix | 3.25 | 1.00 | 153.85 |
| Total | 500 |
The stocks fill the batch: the volumes above add up to 499.7 µL, which is 500 µL within pipetting error. Add no water.
Storage¶
Aliquot Small Molecule Mix into 1.5 mL microfuge tubes (between 50 µL and 100 µL per aliquot) and store at -80 °C.