<aside> <img src="/icons/light-bulb_gray.svg" alt="/icons/light-bulb_gray.svg" width="40px" /> Getting Started

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You want to make PURE. You’ll need Energy Mix, the combination thirty-one (31) small molecules (e.g., rNTPs, amino acids, buffering salts, etc.) and tRNAs (from 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.

<aside> <img src="/icons/merge_gray.svg" alt="/icons/merge_gray.svg" width="40px" /> Prerequisite Protocols

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• [ ] Make Amino Acid Mix (3.25 mM): Make Amino Acid Mix
• [ ] Make tRNAs (40 ug/uL): Make tRNAs

Nucleus_v0.3.0_EMix-worksheet.xlsx

<aside> <img src="/icons/wrench_gray.svg" alt="/icons/wrench_gray.svg" width="40px" /> Materials and Equipment

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					 | $72.46 | 4C to 30C | [[link](<https://us.vwr.com/store/catalog/static_catalog.jsp?catalog_number=97064-756>)] |

| 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 (500 mM; 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 | 4C to 30C | [link] | | Folinic acid | Folinic acid calcium salt hydrate | Sigma-Aldrich | F7878-100MG | $108 | 4C to 30C | [link] | | BME | 2-Mercaptoethanol | Sigma-Aldrich | M6250-100ML | $65.50 | 4C to 30C | [link] | | Spermidine | Spermidine, ≥99% (GC) | Sigma-Aldrich | S2626-5G | $176 | -25C to -15C | [link] | | tRNA | ‣ (50 ug / uL; see Make tRNAs) | | | | -25C to -15C | [link] | | Amino Acids | Amino Acid Mix (3.25 mM; see Make Amino Acid Mix) | | | | -80C to -20C | |

<aside> <img src="/icons/iterate_gray.svg" alt="/icons/iterate_gray.svg" width="40px" /> Protocol

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• [ ] Folinic Acid Preparation
• For more information please see our developer note: An Unexpected Enzyme in PURE: Why Folinic Acid Needs Extra Help

36-Pot PURE

• [ ] Prepare Folinic Acid Stock (2.56 mg / mL, 5 mM).
  • Notes: This step converts Folinic acid to 5,10-methenyl-THF
  • [ ] Prepare 50 mM BME by adding 17.5 uL pure BME (14.3 M) to 50 mL H2O.
  • [ ] Weigh 25 mg Folinic acid.
  • [ ] Resuspend in 2 mL BME (50 mM).
  • [ ] Add 220 uL HCl (1M).
  • [ ] Incubate at 25C / 3 hr. The solution should turn yellow.
  • [ ] Dilute by adding 7.55 mL deionized water and mix by inversion.
  • [ ] Aliquot and freeze at -20C.

One-Pot PURE

• [ ] 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 the following 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

  • Tips: adjust pH then volume

• [ ] Thaw tRNAs on ice immediately before use.

  • Notes: tRNAs are fragile!

• [ ] Combine Energy Mix components.

• [ ] Aliquot Energy Mix into 1.5 mL microfuge tubes (between 50 uL and 100 uL per aliquot) and store at -80C.

<aside> <img src="/icons/book_gray.svg" alt="/icons/book_gray.svg" width="40px" /> Resources and References

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