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

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You have a sample of a biomolecule (e.g., T7RNAP, tRNA, ribosomes, plasmid, etc.) that is either (1) in the wrong buffer (e.g., has 500 mM Imidazole) or (2) is too dilute.

You can use centrifugal spin filters to concentrate your sample and change its buffer. These filters allow buffer and small molecules to flow through them, retaining large molecules bigger than the filter’s specified molecular weight.

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

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We use two different volume spin filters, depending on how much sample volume we need to process: 15 mL or 0.5 mL.

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

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• [ ] Dilute your sample ~10x with your new buffer (e.g., add 54 mL P3 Exchange Buffer to 6 mL eluted protein).
• [ ] Load your sample onto a centrifugal filter column.
  • Notes: use filter cutoffs 3x smaller than your sample.
• [ ] Centrifuge samples at either 4000 rcf (4 mL or 15 mL filters) or 14 000 rcf (0.5 mL filters) at 4C until you’ve reached your target volume. Check on your sample volume in the first 10 min, then again as needed.
  • Notes: if you have too much sample, load in tranches.
• [ ] Continue to dilute and spin your sample, noting your sample volume and dilution volume at each step, until you’ve reached your target dilution factor (we recommend ≥ 300x).
  • Notes: how to calculate dilution factor.
• [ ] Store columns for later use.
  • [ ] Wash columns by loading with ddH2O and spinning.
  • [ ] Load columns with EtOH 20% (v/v) and store at room temp.
• Notes: If you see precipitate, dilute your sample immediately.

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

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• Papers

  • Original PURE paper

  Cell-free translation reconstituted with purified components

  • OnePot PURE

  A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System

  OnePot PURE Cell-Free System | Text Page

<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)

• Developers
  • Murray Lab