Getting Started
You want to make PURE reactions and need to feed them with amino acids. You will need 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.
Materials and Equipment
Name | Product | Manufacturer | Part # | Price | Storage Conditions | Link |
Amino Acids | L-Amino acids, analytical standard | Sigma-Aldrich | LAA21-1KT | $558 | 1C to 4C | [link] |
Potassium Hydroxide | Potassium hydroxide - ACS reagent, ≥85%, pellets | Sigma-Aldrich | 221473-1KG | $99.90 | 4C to 30C | [link] |
Acetic Acid (glacial) | Acetic acid, glacial, ACS reagent, ≥99.7% | Sigma-Aldrich | 695092-100ML | $52.20 | 4C to 30C | [link] |
Protocol
Prepare KOH 0.1 M from stock solution or Potassium Hydroxide pellets
Reagent | MW [g/mol] | Amount [g] | Final Volume [mL] |
Potassium Hydroxide (0.1 M) | 56.11 | 5.61 | 1000 |
Weigh ~ 1.18 mg Tyrosine (Tyr) into a 15 mL conical tube; note mass
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For each amino acid (except Tyrosine; Tyr):
Weigh out between 1 mg and 4 mg; note weight
Transfer to 1.5 mL microfuge tubes and label
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Calculate resuspension volume to bring Tyr to 3.25 mM. Call this volume the “Total Volume”
e.g., 1.18 mg Tyr (MW = 181.2 g / mol) ⇒ 2000 uL resuspension volume
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Given the following target concentrations, calculate resuspension volumes for each of the 19 following amino acid stock solutions
Amino Acid | Stock Concentration (mM) | Molar Weight (g/mol) | Volume Fraction in Final Mix (%) |
Ala | 168 | 89.1 | 1.9% |
Arg | 500 | 210.66 | 0.7% |
Asn | 100 | 132.1 | 3.3% |
Asp | 12.5 | 133.1 | 26.0% |
Cys | 200 | 157.62 | 1.6% |
Glu | 25 | 147.1 | 13.0% |
Gln | 100 | 146.2 | 3.3% |
Gly | 500 | 75.1 | 0.7% |
His | 500 | 209.63 | 0.7% |
Ile | 100 | 131.2 | 3.3% |
Leu | 150 | 131.2 | 2.2% |
Lys | 500 | 182.65 | 0.7% |
Met | 250 | 149.2 | 1.3% |
Phe | 100 | 165.2 | 3.3% |
Pro | 200 | 115.1 | 1.6% |
Ser | 500 | 105.1 | 0.7% |
Thr | 100 | 119.1 | 3.3% |
Trp | 12.5 | 204.2 | 26.0% |
Val | 200 | 117.1 | 1.6% |
Water | - | - | 5.2% |
Given the Total Volume calculated above, and the volume fractions given above, calculate how much of each amino acid solution to add to the final amino acid mix.
Confirm that you will have enough volume of each stock solution to make the final mix (else, go weigh more amino acids and recalculate)
Resuspend each the following amino acids in milliQ water to the volumes that you calcualted: Ala, Arg, Asn, Glu, Gln, Gly, Pro, Ser, Thr, Val
Resuspend each of the following amino acids in 0.1 M KOH: Asp, Cys, His, Ile, Leu, Lys, Met, Phe, Trp, Tyr
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Combine each stock solution in the 15 mL conical tube containing Tyr. Resuspend by vortexing
Titrate combined stock to pH=7.6 using glacial acetic acid (should be a tiny amount)
Finish amino acid mix by adding water to final volume
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Filter amino acid mix (0.22 um)
Aliquot amino acid mix into 1.5 mL microfuge tubes (between 200 uL and 500 uL per aliquot)
Store amino acid stock solutions and amino acid mix aliquots frozen (between -15C and -85C)
Resources and References
Other Protocols
The OnePot PURE methods paper(JOVE) has a great interactive table!
- Use Supplementary Table 3 for making Amino Acid Mix.
Papers
Original PURE paper
OnePot PURE
Resources
Credits
Developers
Testers