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

Microfluidic electrophoresis is a technique to separate different-sized DNA fragments within microcapillaries.

To begin, take a multi-well microfluidic chip having designated wells for the mixture of gel-based polymer and fluorescent dye, DNA samples, and a known molecular-weight DNA ladder. These wells are interconnected via a network of microchannels.

Dispense gel-dye mix into allocated wells. Assemble the chip on a priming station pre-fitted with an empty syringe. Press down the syringe plunger, applying pressure to force the gel-dye mix into the separation microchannel.

Remove the chip and pipette other components into their respective wells. Add a marker - an internal standard that calibrates the sample sizing - to the wells containing the ladder and samples.

Insert the microchip into a fragment analyzer to run the chip. Once inside, the applied voltage generates electrophoretic forces, driving the sample from the well into the sample-loading microchannel from where it reaches the cross-intersection between this channel and the separation channel. The electric current helps the sample enter the separation microchannel.

The fluorescent dye molecules intercalate negatively charged DNA fragments which move toward the anode at different electrophoretic velocities relative to their molecular size - smaller fragments migrating faster than larger ones. Consequently, the fragments separate into different-sized bands that fluoresce.

A laser-based detector measures each band's fluorescence and records it as an electropherogram that displays different-sized DNA as a distinct peak.

Prior to starting, bring the DNA dye concentrate, DNA gel matrix, DNA marker, DNA ladder, and purified DNA samples to room temperature. Set up the priming station by replacing the syringe, and adjusting the base plate. Then, release the lever of the syringe clip and slide it to the top position.

Start the sizing software and prepare the gel-dye mix. Vortex the dye concentrate for 10 seconds, and spin it down. Then, add 25 microliters of the dye to a gel matrix vial, and vortex the solution to mix.

Transfer the gel-dye mix to a spin filter. Place it in the centrifuge, and spin it for 10 minutes at 1,500 times g. When ready to load the gel-dye mix, insert a new DNA chip into the priming station, and add 9 microliters of the gel-dye mix into the well that is marked with "G".

Close the priming station, and ensure that the plunger is positioned at the 1-milliliter mark. Press the syringe plunger down, until it is held by the clip. Wait for exactly 30 seconds, and then release the clip. Wait for 5 more seconds, and then slowly pull the plunger back to the 1-milliliter position.

Open the priming station, and add 9 microliters of gel-dye mix into the wells marked with "G". Add 5 microliters of marker into the well marked with a ladder symbol, and each of the 12 sample wells.

Add 1 microliter of the ladder to the well with the ladder symbol, and 1 microliter of the PCR product or water to the sample wells. Vortex the chip for 1 minute to 2,400 RPM. Insert it into the bioanalyzer, and run the chip within five minutes.