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University of Wisconsin–Madison

DNA Sequencing Facility

Sequencing Materials and Methods

Typical sequencing reactions consisted of 2 ul of BigDye Terminator v. 3.1 mix (Applied Biosystems), 3 ul of dilution buffer (Applied Biosystems), 5-20 pmol of primer, and 0.2 ug of template DNA in a final reaction volume of 20 ul.  Cycle conditions were an initial denaturation at 96° for 2’, then 35 cycles of 96° for 10”, 52° for 15”, 60° for 3', followed by 1’ at 72°.  Excess dye terminators were removed using CleanSeq magnetic bead sequencing reaction clean up kit from Beckman Coulter(Agencourt Bioscience).  The samples were resuspended off of the beads in 50 ul of ddH2O.  10 ul of each sample was loaded into a 96 well PCR plate, an additional 10 ul of ddH2O was added to each well, and the plate loaded onto the sequencers according to the manufacturer's instructions.   Samples were electrophoresed on an Applied Biosystems 3730xl automated DNA sequencing instrument, using 50 cm capillary arrays and POP-7 polymer.  Data were analyzed using PE-Biosystems version 3.7 of Sequencing Analysis

Unless otherwise specified, all data and reagents distributed by the University of Wisconsin Biotechnology Center DNA Sequencing Facility are intended for research purposes only. They are not intended nor certified for diagnostic or clinical use.  Clinical services are provided through our collaboration with the UW Collaborative Genomics Core.