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
- 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 diluted PCR product. Seal, mix and centrifuge briefly.
CAUTION: Handle with care. Mixing formamide with water generates formic acid, which is toxic. - 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.
- 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.
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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 |