Capillary Electrophoresis: A Technique to Analyze Fluorescently Labeled Polymerase Chain Reaction Amplicons

Abstract

Source: Gulla, S. et al., Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis. J. Vis. Exp. (2019)

This video describes the technique of capillary electrophoresis to analyze fluorescently labeled PCR amplicons obtained from various samples based on their size. It generates a migratory pattern that determines the fragments’ base-pair lengths with the help of the labeled fluorescent dye in each fragment.

Protocol

1. Capillary Electrophoresis Setup and Run Conditions Following confirmation of PCR amplicons, dilute PCR products 1:10 (v/v) in purified water. Seal, mix and centrifuge briefly. Working in a fume cupboard, prepare a volume of master mix consisting of 9 µL of formamide and 0.5 µL of size standard per PCR product (allow 10% surplus volume). Vortex briefly and distribute 9.5 µL into wells on a plate appropriate for the available CE system before adding 0.5 µL of di…

Materials

Avant 3500xL Genetic Analyzer	Thermo Fisher Scientific	A30469	Used for capillary electrophoresis fragment analysis.
GelRed Nucleic Acid Stain, 10,000X in water	Biotium	41003	Fluorescent nucleic acid dye used during gel electrophoresis.
GeneMapper Software 5	Thermo Fisher Scientific	4475073	Used for reading electropherograms from capillary electrophoresis.
GeneScan 600 LIZ dye Size Standard v2.0	Thermo Fisher Scientific	4408399	Size standard used during capillary electrophoresis.
Hi-Di Formamide	Thermo Fisher Scientific	4311320/4440753	Deionized formamide used during capillary electrophoresis. Prepare working aliquouts.
Milli-Q water	NA	NA	Purified water used during PCR and capillary electrophoresis. Standard recipe; produced inhouse.
POP-7 Polymer for 3500 Dx/3500xL Dx Genetic Analyzers	Thermo Fisher Scientific	A26077/4393713/4393709	Separation matrix used during capillary electrophoresis.
UV-based gel imaging/visualisation system	As preferred.	NA
Vortexer	As preferred.	NA