Microfluidic Capillary Electrophoresis for Fragment Sizing of PCR Products: A Microchip-Based Electrophoretic Separation Technique to Separate DNA Fragments Based on Size

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

1. 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.
2. Set up the priming station
   1. Replace the syringe (see Table of Materials) when using a new batch of reagents.
   2. Adjust the base plate, and release the lever of the syringe clip and slide it up to the top position.
3. Start the sizing software (see Table of Materials) and prepare the gel-dye mix.
4. Vortex dye concentrate for 10 s and spin down. Add 25 μL of the dye to a gel matrix vial. Vortex the mixed solution well and spin down.
   1. Transfer the gel-dye mix to a spin filter. Place the spin filter in a microcentrifuge and spin for 10 min at room temperature at 1,500 x g ± 20%.
      NOTE: Protect the solution with dye from light and store at 4 °C after use. The gel-dye mix can be used for about 15 chips once prepared. Allow the gel-dye mix to equilibrate to room temperature for 30 min each time before use.
5. Load the gel-dye mix
   1. Insert a new DNA chip on the priming station. Add 9 μL of gel-dye mix into the well marked with “G”. Please ensure the plunger is positioned at the 1 mL mark and then close the priming station.
   2. Press the syringe plunger down until it is held by the clip. Wait for exactly 30 s then release the clip. Wait for 5 s, and then slowly pull the plunger back to the 1 mL position.
   3. Open the priming station and add 9 μL of gel-dye mix into the wells marked with “G”.
6. Add 5 μL of marker into the well marked with the ladder symbol and also add 5 μL of marker into each of the 12 sample wells. Do not leave any wells empty.
7. Add 1 µL of DNA ladder into the well marked with the ladder symbol. Add 1 µL of PCR product (used wells) or 1 µL of ultrapure water (unused wells) into each of the 12 sample wells. Put the chip horizontally in the adapter of vortex mixer and vortex for 1 min at the indicated setting (2,400 rpm).
8. Insert the chip in the bioanalyzer instrument and run the chip in the instrument within 5 min.
9. After the assay is complete, immediately remove the used chip from the instrument.
10. Slowly add 350 μL of deionized water into one of the wells of the electrode cleaner. Open the lid of the bioanalyzer and place the electrode cleaner into it. Close the lid and incubate for about 10 s. Open the lid and remove the electrode cleaner. Wait another 10 s to allow the water on the electrodes to evaporate and then close the lid.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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