Overview¶
Small Molecule Mix 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 Small Molecule Mix, 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.
Notes
None
Prerequisite Documentation
Hazardous Materials
None
Critical Materials
| Reagent | Product Name | Manufacturer | Part # | Price | Storage Conditions | Link |
|---|---|---|---|---|---|---|
| Folinic acid | Folinic acid calcium salt hydrate | Sigma-Aldrich | F7878-100MG | $108 | 4C to 30C | [link] |
Genetically Encoded Components
None
Composition
| Reagent | Concentration in Small Molecule Mix (mM) | Concentration in Final Reaction (mM) |
|---|---|---|
| HEPES-KOH (pH 7.6) | 125 | 50 |
| Potassium glutamate | 250 | 100 |
| Magnesium acetate | 18.75 | 7.5 |
| rATP | 5 | 2 |
| rGTP | 5 | 2 |
| rCTP | 2.5 | 1 |
| rUTP | 2.5 | 1 |
| Amino Acids (each) | 0.75 | 0.3 |
| Creatine phosphate | 50 | 20 |
| Folinic acid | 0.05 | 0.02 |
| Spermidine | 5 | 2 |
| TCEP | 2.5 | 1 |
References
None
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 -20C.
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 | 4C to 30C | no | no |
| HEPES-KOH (pH 7.6, 1 M) | 238.3 | 59.5 | 250 | 4C to 30C; dark | yes | no |
| Potassium glutamate (2.5 M) | 203.23 | 21.8 | 50 | 4C to 30C | no | no |
| Magnesium acetate (1 M) | 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 | See above | -25C to -15C | no | no | |
| Spermidine (500 mM) | 145.25 | 1 | 13.77 | -25C to -15C | no | no |
| Amino Acid Mix | see Make Amino Acid Mix | -85C to -15C | 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 | 125 | 62.5 |
| Potassium glutamate | 2500 | 250 | 50.0 |
| Magnesium acetate | 1000 | 18.75 | 9.38 |
| rATP | 100 | 5 | 25.0 |
| rGTP | 100 | 5 | 25.0 |
| rCTP | 100 | 2.5 | 12.5 |
| rUTP | 100 | 2.5 | 12.5 |
| 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 Mix | 3.25 | 0.75 | 115.4 |
| Ultrapure water | n/a | n/a | 15.82 |
| Total | 500 |
Storage¶
Aliquot Small Molecule Mix into 1.5 mL microfuge tubes (between 50 uL and 100 uL per aliquot) and store at -80C.