Sequencing BAC DNA With Near-Infrared
Fluorescent Non-Nucleotide Terminators

Stephen C. Roemer, Vince R. Boveia, Craig M. Johnson and D. Michael Olive
LI-COR Inc., Biotechnology, 4647 Superior Street, Lincoln, NE 68504

Materials and Methods

IRD800-AcyATP

BAC Sequencing Protocol Note: Avoid vortexing the samples DNA samples should be stored at 4°C prior to sequencing. Sequencing mixture: DNA template: 1.5-2.0 µg Thermo Sequenase®: 8 units LI-COR AcyNTP mixtures: 2 µl/rx Water: to 27 µl Thermal Cycling IRDye® infrared dye AcycloTerminators 95 °C for 3 minutes 95 °C for seconds 50 °C for seconds 72 °C for 2 minutes Repeat 2 to 4 for 60 cycles total 72 °C for 5 minutes 4 °C soak Labeled Primers 95 °C for 3 minutes 95 °C for 30 seconds 50 °C for 30 seconds 70 °C for 2 minutes Repeat 2 to 4 for 60 cycles total 4 °C soak