Capillary electrophoresis fragment analysis

The Facility operates an Applied Biosystems 3500 Genetic Analyser; an 8-capillary system capable of fragment analysis for a range of projects from single sequencing reads to 96 well plates of microsatellite or AFLP genotyping products.

For the analysis of pre-sequenced samples

Users perform their own sequencing reactions using Big Dye reagents, and supply the dried down reactions in 8-strip tube or a 96-well plate format ready for resuspension and electrophoresis on the ABI3500

For fragment analysis of PCR-amplified microsatellite or AFLP samples

Fluorescently labelled PCR-amplified microsatellite or AFLP products (up to 96 samples at a time) are supplied to the facility in 8-strip tube or a 96-well plate format diluted ready for electrophoresis on the ABI3500. The instrument is capable of detecting and separating the emissions of up to five dyes of different colours in a single experiment, and is calibrated to detect dye set 33 (DS-33)

DS-33: 6-FAM, VIC, PET, and NED and LIZ (LIZ is usually reserved for the size standard)

For microsatellite genotyping/fragment analysis, 5’ end labeled forward primers and unlabeled reverse primers can be used for amplifying the genomic repeat region using a suitable fluorescent dye label from this dye set.

Note: 6-FAM gives the strongest signal and should be used if there is no preference.   Samples labelled with different fluorescent dye labelled primers from the same dye set can be pooled and run together in a single well.

Capillary electrophoresis sequencing and fragment analysis.

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