Automated Capillary Electrophoresis Based Fragment Size Distribution Analysis of cfDNA: A DNA Microchip-based Method to Study Size Distribution of DNA Fragments Isolated from Biofluids
In this video, we analyze the size distribution of cell-free DNA or cfDNA fragments using automated capillary electrophoresis. This technique helps gain insights into the profile of DNA fragments released by cancer cells during apoptosis.
Protocol
1. DNA Fragment Size Distribution of cfDNA by Fragment Analyzer The step before initiating the procedure: Equilibrate the DNA dye concentrate and DNA gel matrix to room temperature for 30 min. Preparation of the gel-dye mix NOTE: Handle solutions with caution as DMSO is known to facilitate the entry of organic molecules into tissues. Thoroughly thaw the DMSO by vortexing the DNA dye concentrate vial for 10 s. Pipette out 15 µL of this concent…
Divulgaciones
The authors have nothing to disclose.
Materials
2100 Bioanalyzer Instrument
Agilent Technologies, Inc.
G2939BA
The 2100 Bioanalyzer system is an established automated electrophoresis tool for the sample quality control of biomolecules.
Adjustable Clip for Priming Station
Agilent Technologies, Inc.
5042-1398
Used in combination with syringe to apply defined pressure for chip priming.
cf-DNA/cf-RNA Preservative Tubes
Norgen Biotek Corp.
63950
Norgen's cf-DNA/cf-RNA preservative tubes are closed, evacuated plastic tubes for the collection and the preservation of cf-DNA, circulating tumor DNA, cf-RNA and circulating tumor cells in human whole blood samples during storage and shipping
Chip Priming Station
Agilent Technologies, Inc.
5065-4401
Used to load gel matrix into a chip with a syringe provided with each assay kit— used for RNA, DNA, and protein assays. Includes priming station, stop watch, and 1 syringe clip
Electrode Cleaner Kit
Agilent Technologies, Inc.
5065-9951
Prevents cross-contamination. Removes bacterial or protein contaminants from electrodes.
Filters for Gel Matrix
Agilent Technologies, Inc.
185-5990
Used for proper mixing of DNA dye concentrate and DNA gel matrix
IKA Basic Chip Vortex
IKA-Werke GmbH & Co. KG
MS-3-S36
Used for proper mixing of DNA ladder and DNA sample on Bioanalyzer assay chips
50 ml centrifuge tubes
Ethanol (96–100%)
Heating block or similar at 56 °C (capable of holding 2 ml collection tubes)
Phosphate-buffered saline (PBS)
Pipettes (adjustable)
Sterile pipette tips (pipette tips with aerosol barriers are recommended to help prevent cross-contamination)
Water bath or heating block capable of holding 50 mL centrifuge tubes at 60 °C
Automated Capillary Electrophoresis Based Fragment Size Distribution Analysis of cfDNA: A DNA Microchip-based Method to Study Size Distribution of DNA Fragments Isolated from Biofluids. J. Vis. Exp. (Pending Publication), e20672, doi: (2023).