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Encyclopedia of Experiments: Biological Techniques

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Microfluidic Capillary Electrophoresis for Fragment Sizing of PCR Products


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.

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