Make Amino Acid Mix

Overview¶

Amino Acid Mix is an equimolar (3.25 mM) mix of the 20 canonical amino acids. Here, we make stock solutions for each amino acid and assemble them into Amino Acid Mix for use in downstream protocols. The assembly worksheet is adapted, with edits, from OnePot PURE (2019).

Materials and Equipment¶

Protocol¶

Prepare Amino Acid Stock Solutions.¶

• Use the included Amino Acid Mix worksheet included as a resource for this protocol to calculate required masses and volumes.

• Weigh Tyr into the cap of a 15 mL conical tube. Note mass and recalculate required final volume of AA mix to bring Tyr to 3.25 mM.

• Label 19x microfuge tubes, one for each amino acid other than Tyr.

• Weigh each amino acid into a separate tube and note mass. Make sure reading stabilizes before noting.

• Check if resuspension volume will fit assigned tube. Else, adjust mass.

• Filter ultrapure water (0.22 µm) and add the corresponding calculated resuspension volume to each tube of powdered amino acids.

• Resuspend 19x amino acid stocks (excluding Tyr).

  • Put each microfuge tube on a heater shaker set to 65°C / 1400 rpm.

  • Check for complete resuspension every 30 min (should take ≤ 1 hr). Keep stocks at 65°C for next step.

Assemble Amino Acid Master Mix.¶

• Put Tyr 15 mL tubes in hot water bath sonicator set to 65°C. Keep other 19x amino acid stocks on heater shaker at 65°C.

• For each amino acid stock other than Tyr, add the calculated volume to Tyr 15 mL tube. Keep the Tyr tube in 65°C sonicator bath to maintain temperature and facilitate resuspension.

• Add filtered ultrapure water to final concentration as specified.

• Resuspend amino acid mix in hot water bath sonicator set to 65°C with sonication.

  • Note incubation start time:

  • Check for complete resuspension every 30 min (should take ~ 30 min).

• Syringe filter using 0.22 µm filter.

Storage¶

• Aliquot and store at -20°C.

Downloads¶