Capillary Electrophoresis: A Technique to Analyze Fluorescently Labeled Polymerase Chain Reaction Amplicons
Transcript
Following confirmation of PCR amplicons, use new PCR strips or plates, to dilute PCR products 1 to 10 in purified water. Vortex and centrifuge briefly. While working in a fume cupboard, prepare a master mix in a centrifuge tube consisting of formamide and reference size standard solution.
According to the number of PCR products to be analyzed, use reagent volumes, as detailed in the manuscript. Allow a 10% surplus volume.
Vortex briefly and distribute 9.5 microliters into individual wells on a PCR plate, appropriate for the available capillary electrophoresis system. Then, add 0.5 microliters of diluted PCR product. Seal and centrifuge briefly.
Then, load the plate containing the samples into a PCR thermal cycler, and denature the samples at 95 degrees Celsius for 3 minutes, before cooling to 4 degrees Celsius indefinitely. Centrifuge at 1,000 x g for 1 minute. Carefully remove the seal, and load the plate onto a calibrated capillary electrophoresis system according to the manufacturer's instructions.
Run fragment analysis capillary electrophoresis using reagents as appropriate for the apparatus of choice, and adjust the run conditions according to the description in the manuscript.
