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

  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.

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

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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).

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