<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, which will need amino acids. You will need to make Amino Acid Mix. Here, we make stock solutions for each amino acid, plus an equimolar (3.25 mM) master mix of amino acids for use in downstream protocols.
Nucleus_v0.3.0_AA_worksheet.xlsx
<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 |
|---|---|---|---|---|---|---|
| Amino Acids | L-Amino acids, analytical standard | Sigma-Aldrich | LAA21-1KT | $558 | 1C to 4C | [link] |
<aside> <img src="/icons/iterate_gray.svg" alt="/icons/iterate_gray.svg" width="40px" /> Protocol
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[ ] Using the included AA mix worksheet at the top of this page, determine the minimum mass you will need for each amino acid given how much amino acid mix you want to make
Tips: weigh aminos, then adjust volume.
Notes: ≥ 4 mg is enough for ~5 mL AA mix
[ ] For each amino acid: tare a microcentrifuge tube (1.5 mL) on an analytical balance, add at least the minimal mass you determined previously, and input the actual mass weighed in the worksheet.
[ ] For each amino acid, resuspend in Ultrapure water (18.2 MOhm, e.g., Milli-Q) with "calculated resuspension volume" in the worksheet.
[ ] For each amino acid, note the volume to add to the final amino acid mix. Transfer this volume of each stock to a 15 mL conical tube.
[ ] Add Ultrapure water to amino acid mix to reach the target final volume and directly add Tyrosine to the final volume of amino acid mix. Tyrosine is the least soluble amino acid, and therefore, a larger volume is required to solubilize tyrosine.
[ ] Filter amino acid mix with a syringe filter (0.22 um).
[ ] Aliquot amino acid mix into 1.5 mL microcentrifuge tubes (between 200 uL and 500 uL per aliquot)
[ ] Store amino acid stock solutions and amino acid mix aliquots frozen (between -15C and -85C)
<aside> <img src="/icons/book_gray.svg" alt="/icons/book_gray.svg" width="40px" /> Resources and References
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The OnePot PURE methods paper (JOVE) has a great interactive table! Use Supplementary Table 3 for making Amino Acid Mix. We based our worksheet on this one.