Make Amino Acid Mix

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.

Nucleus_v0.2.1_AA_worksheet.xlsx12.1KB

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

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 → 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, note the calculated resuspension volume in the worksheet.

Resuspend Ala, Arg, Asn, Glu, Gln, Gly, Pro, Ser, Thr, Val in milliQ water to the calculated resuspension volumes.

‣

Tips: vortex, heat, and/or sonciate stocks to aid resuspension

Resuspend Asp, Cys, His, Ile, Leu, Lys, Met, Phe, Trp, Tyr in 0.1 M KOH to the calculated resuspension volumes.

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 by pipetting.

Titrate combined stock to pH=7.6 using glacial acetic acid.

Add milliQ water to amino acid mix to reach the target final volume

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

The OnePot PURE methods paper (JOVE) has a great interactive table! Use Supplementary Table 3 for making Amino Acid Mix.

OnePot-JOVE-Table-S3-amino-acid-worksheet.xlsx12.3KB

Papers

Original PURE paper

Cell-free translation reconstituted with purified components

Nature Biotechnology - Cell-free translation reconstituted with purified components

doi.org

Cell-free translation reconstituted with purified components

OnePot PURE

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

We demonstrate a simple, robust, and low-cost method for producing the PURE cell-free transcription–translation system. Our OnePot PURE system achieved a protein synthesis yield of 156 μg/mL at a cost of 0.09 USD/μL, leading to a 14-fold improvement in cost normalized protein synthesis yield over existing PURE systems. The one-pot method makes the PURE system easy to generate and allows it to be readily optimized and modified.

doi.org

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

OnePot PURE Cell-Free System | Text Page

Scientific Article | The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for...

dx.doi.org

OnePot PURE Cell-Free System | Text Page

Credits

Developers