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…
Divulgaciones
The authors have nothing to disclose.
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.