Microfluidic Capillary Electrophoresis for Fragment Sizing of PCR Products: A Microchip-Based Electrophoretic Separation Technique to Separate DNA Fragments Based on Size
Published: April 30, 2023
Abstract
Source: Wang, H., Zhu et al. A Robust Polymerase Chain Reaction-based Assay for Quantifying Cytosine-guanine-guanine Trinucleotide Repeats in Fragile X Mental Retardation-1 Gene. J. Vis. Exp. (2019).
This video describes the microfluidic capillary electrophoresis technique to separate PCR amplicons based on their size. This technique helps in fragment sizing using fluorescent dyes and standard molecular size markers.
Protocol
1. Fragment Sizing of PCR Products Prior to starting, allow DNA dye concentrate, DNA gel matrix, DNA marker, DNA ladder and purified DNA samples to equilibrate to room temperature for 30 min. Set up the priming station Replace the syringe (see Table of Materials) when using a new batch of reagents. Adjust the base plate, and release the lever of the syringe clip and slide it up to the top position. Star…
Declarações
The authors have nothing to disclose.
Materials
| Agilent 2100 Bioanalyzer instrument: 0.2 mL PCR tubes | Axygen | PCR-02D-C | |
| Agilent 2100 Bioanalyzer instrument: 1x TE buffer, pH 8.0, Rnase-free | Ambion | AM9849 | |
| Agilent 2100 Bioanalyzer instrument: 2100 Bioanalyzer instrument | Agilent | G2939AA | |
| Agilent 2100 Bioanalyzer instrument: 96-well PCR Plate | Thermo Fisher | AB0800 | |
| Agilent 2100 Bioanalyzer instrument: Electrode cartridge | Agilent | Supplies equipment of the 2100 Bioanalyzer instrument | |
| Agilent 2100 Bioanalyzer instrument: IKA vortex mixer | Agilent | Supplies equipment of the 2100 Bioanalyzer instrument | |
| Agilent 2100 Bioanalyzer instrument: Sizing software 2100 Expert software | Agilent | Supplies equipment of the 2100 Bioanalyzer instrument | |
| Agilent 2100 Bioanalyzer instrument: Test chips | Agilent | Supplies equipment of the 2100 Bioanalyzer instrument | |
| Agilent DNA 7500 kit | Agilent | 5067-1506 | For Fragment sizing |
| Agilent DNA 7500 kit: DNA 7500 Ladder (yellow cap) | Agilent | In kit: Agilent DNA 7500 kit (catalog number: 5067-1506) | |
| Agilent DNA 7500 kit: DNA 7500 Markers (green cap) | Agilent | In kit: Agilent DNA 7500 kit (catalog number: 5067-1506) | |
| Agilent DNA 7500 kit: DNA chips | Agilent | In kit: Agilent DNA 7500 kit (catalog number: 5067-1506) | |
| Agilent DNA 7500 kit: DNA Dye Concentrate (blue cap) | Agilent | In kit: Agilent DNA 7500 kit (catalog number: 5067-1506) | |
| Agilent DNA 7500 kit: DNA Gel Matrix Vial (red cap) | Agilent | In kit: Agilent DNA 7500 kit (catalog number: 5067-1506) | |
| Agilent DNA 7500 kit: Electrode Cleaner | Agilent | In kit: Agilent DNA 7500 kit (catalog number: 5067-1506) | |
| Agilent DNA 7500 kit: Spin Filter | Agilent | Supplies of Agilent DNA 7500 kit (catalog number: 5067-1506) | |
| Agilent DNA 7500 kit: Syringe | Agilent | Supplies of Agilent DNA 7500 kit (catalog number: 5067-1506) | |
| Chip priming station | Agilent | 5065-4401 | Supplies equipment of the 2100 Bioanalyzer instrument |
| Filter plate vacuum Manifold: MultiScreenHTS Vacuum Manifold | Merck Millipore | MSVMHTS00 | Vacuum instrument for Filter plate vacuum Manifold for PCR product purification |
| Filter plate vacuum Manifold: Silicone stopper | Merck Millipore | XX2004718 | Filter plate vacuum Manifold |
| Filter plate vacuum Manifold: Vacuum pump | Merck Millipore | WP6122050 | Filter plate vacuum Manifold |
| Filter plate vacuum Manifold: Waste collection vessel | Merck Millipore | XX1004705 | Filter plate vacuum Manifold |
Citar este artigo
Microfluidic Capillary Electrophoresis for Fragment Sizing of PCR Products: A Microchip-Based Electrophoretic Separation Technique to Separate DNA Fragments Based on Size. J. Vis. Exp. (Pending Publication), e21115, doi: (2023).