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A low-cost and open-source protocol to produce key enzymes for molecular detection assays.

STAR protocols (2021-11-13)
Gabriel Mendoza-Rojas, Vanessa Sarabia-Vega, Ana Sanchez-Castro, Lesia Tello, Luis Cabrera-Sosa, Jose A Nakamoto, Katherin Peñaranda, Vanessa Adaui, Roberto Alcántara, Pohl Milón
ABSTRACT

Here, we describe a detailed step-by-step protocol for the expression, purification, quantification, and activity determination of key enzymes for molecular detection of pathogens. Based on previous reports, we optimized the protocol for LbCas12a, Taq DNA polymerase, M-MLV reverse transcriptase, and TEV protease to make it compatible with minimal laboratory equipment, broadly available in low- and middle-income countries. The enzymes produced with this protocol have been successfully used for molecular detection applications. For complete details on the use and execution of this protocol, please refer to Alcántara et al. (2021a, 2021b).

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Piperidine, ReagentPlus®, 99%
Millipore
NP-40 Alternative, A non-ionic surfactant used in the isolation and purification of functional membrane protein complexes and in two-dimensional (2D) gel electrophoresis.
Sigma-Aldrich
Protein standard, liquid, 2 mg protein/ml
Millipore
EDTA, Disodium Salt, Dihydrate, Molecular Biology Grade - CAS 6381-92-6 - Calbiochem