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A simple and affordable kinetic assay of nucleic acids with SYBR Gold gel staining.

ABSTRACT: Labeling substrates or products are paramount in determining enzymatic kinetic parameters. Several options are available; many laboratories use either radioactive or fluorescent labeling because of their high sensitivity. However, those methods have their own drawbacks such as half-life decay, expensive and hazardous. Here, we propose a novel, simple, economical and fast alternative to substrate labeling for studying the kinetics of nucleic acids: post-migration gel staining with SYBR Gold. Cleavage rates similar to the ones reported in the literature for the I-R3 DNA-cleaving DNA enzyme in the presence of zinc chloride are an indication of the quality of the new method. Moreover, the activity of the hammerhead ribozyme was also monitored by our method to illustrate its versatility. This labeling-free method has several advantages such as its ease of use as well as cost effective and versatility with both non-structured and structured RNAs or DNAs.

SUBMITTER: Guillen D

PROVIDER: S-EPMC7053750 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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A simple and affordable kinetic assay of nucleic acids with SYBR Gold gel staining.

Guillen Danielle D Schievelbein Mika M Patel Kushkumar K Jose Davis D Ouellet Jonathan J

PloS one 20200303 3

Labeling substrates or products are paramount in determining enzymatic kinetic parameters. Several options are available; many laboratories use either radioactive or fluorescent labeling because of their high sensitivity. However, those methods have their own drawbacks such as half-life decay, expensive and hazardous. Here, we propose a novel, simple, economical and fast alternative to substrate labeling for studying the kinetics of nucleic acids: post-migration gel staining with SYBR Gold. Clea ...[more]

PMID: 32126098

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