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

Overview

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

1. Following confirmation of PCR amplicons, dilute PCR products 1:10 (v/v) in purified water. Seal, mix and centrifuge briefly.
2. 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 diluted PCR product. Seal, mix and centrifuge briefly.
   CAUTION: Handle with care. Mixing formamide with water generates formic acid, which is toxic.
3. Using a PCR thermal cycler, denature the samples at 95 °C for 3 min before cooling to 4 °C indefinitely. Centrifuge briefly and load the plate onto a calibrated CE system according to the manufacturer’s instructions.
4. Run fragment analysis CE using reagents as appropriate for the apparatus of choice and the following settings: 60 °C; 5 s injections at 1.6 kV (32 V per cm); 32 min run time at 15 kV (300 V per cm). CE fragment analysis of 24 wells on a 24-capillary (50 cm) will typically take approximately 50 min.

Materials

Name	Company	Catalog Number	Comments
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