<aside> <img src="/icons/light-bulb_gray.svg" alt="/icons/light-bulb_gray.svg" width="40px" /> Getting Started
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You have proteins and want to store them at -80C until you’re ready to use them. Here is how you do that.
<aside> <img src="/icons/wrench_gray.svg" alt="/icons/wrench_gray.svg" width="40px" /> Materials and Equipment
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| 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] |
| Magnesium Chloride hexahydrate | BioXtra Magnesium chloride, Hexahydrate, Powder or Crystals, ≥99.0% | Sigma-Aldrich | M2670-100G | $50.50 | 4C to 30C | [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 | 4C to 30C | [link] |
| Glycerol | Glycerol, Molecular Biology Grade, Liquid, ≥99.0% | Sigma-Aldrich | G5516-1L | $157.00 | 4C to 30C | [link] |
| 10 mL Luer lock syringes | Syringes with BD Luer-Lok® Tip, BD Medical, Syringe with Luer-Lok® Tip, Volume=10 mL | BD Industrial/Difco | 550-80620-PK | $102.40 | 4C to 30C | [link] |
| 0.22 um syringe filters | PES Syringe Filter, 0.22μm, 30mm, Sterile | CELLTREAT Scientific Products | 229747 | $84 | 4C to 30C | [link] |
| -80C Freezer | TSX Series Ultra-Low Freezers | Thermo Scientific | TSX60086A | unlisted | 4C to 30C | [link] |
<aside> <img src="/icons/iterate_gray.svg" alt="/icons/iterate_gray.svg" width="40px" /> Protocol
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[ ] Prepare Stock Solutions
| Reagent | MW [g/mol] | Amount [g] | Final Volume [mL] | Storage | Needs pH adjustment? | Needs Sterilization? |
|---|---|---|---|---|---|---|
| Stock Solutions | ||||||
| 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 |
| Magnesium Chloride (1M) | 203.3 | 20.3 | 100 | 4C to 30C | no | no |
[ ] Make 4x P3 Concentrate - used to prepare P3 Exchange and P3 Storage Supplement.
[ ] Make P3 Storage Supplement - used to protect samples in P3 Exchange buffer from damage by freezing during storage at -80C. Add an equal volume of P3 Storage Supplement to samples in P3 Exchange Buffer (1:1). Filter P3 Storage Supplement with a 0.22 um syringe filter.
| Reagent | Final Concentration [mM] |
|---|---|
| HEPES-KOH | 50 |
| Potassium Chloride | 100 |
| Magnesium Chloride | 10 |
| TCEP | 1 |
| Glycerol | 60% (v/v) |
| Reagent | Volume to Add [mL] |
|---|---|
| 4x P3 Concentrate | 2.5 |
| Glycerol | 6.0 |
| TCEP (0.5 M) | 0.020 |
| water (milliQ) | 1.48 |
| Total | 10 |
<aside> <img src="/icons/book_gray.svg" alt="/icons/book_gray.svg" width="40px" /> Resources and References
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Papers
Cell-free translation reconstituted with purified components
A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System
<aside> <img src="/icons/megaphone_gray.svg" alt="/icons/megaphone_gray.svg" width="40px" /> Credits
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Yan Zhang, Zoila Jurado, and Miki Yun (Richard Murray Lab, Caltech)