Pulsed-field gel electrophoresis, which alternates periodically between two differently oriented electric fields, allows the separation of DNA molecules up to 5 Mb in length, and works on the principle that the time it takes for a DNA molecule to respond to an alternatingly oriented electric field depends on its size. Smaller molecules reorient themselves faster and therefore move faster through the gel, so that molecules of different sizes are successfully separated.
Operation method
inverse electric field
Materials and Instruments
DNA Move 1. Prepare a 1% agarose gel for horizontal electrophoresis by placing it in the electrophoresis apparatus and covering it with 2-3 mm of GTBE or TBE buffer.2. Add liquid samples. Set up a peristaltic pump to circulate the electrophoresis buffer. 3. For more product details, please visit Aladdin Scientific website.
GTBE TBE Agarose
Electrophoresis
3. Adjust the cycling rate to 5~10 ml/min for microgels and 20~50 ml/min for large gels.
4. Connect the end of the tube to the recirculation port in the gel tank or directly into the buffer tank.5. Connect the programmable converter to a constant voltage DC power supply, then connect the gel unit to the converter (see table below) and start electrophoresis until the bromophenol blue migrates 1 cm, turn on the converter and peristaltic pump.6. Ethidium bromide staining was performed and photographed as for standard agarose gels. After acid treatment to depurinate the DNA, the gel is ready for Southern hybridization.
(1) These equations assume the use of 0.5xTBE buffer; for GTBE and TAE buffers, the isolated fragments will be slightly larger and electrophoresed about 20% and 30% faster, respectively.(2) Variables: T, temperature (°C); V, electric field flux (V/cm); A, percent agarose concentration (for pulsed-field level agarose multiplied by 0.8); pulse time (for inverted-field gels refers to the reverse time in s); R, ratio of forward to reverse electrophoresis time; θ, angle of reorientation.
(3) Inverted electric field gels do not separate lengths outside this range, and reorientation angle gels can separate fragments outside this range, and not very well.