Department of Pharmaceutics
School of Pharmacy
Center for DNA Sequencing and Gene Analysis
H260 Health Sciences Center
Box 357610
Seattle, WA 98195 -7610
(206) 616-8718
Fax: (206) 543-3204
BigDye Sequencing SOP Version 1.0
1. Each sample should contain template and primer (~5 pmol) in a total volume of 10 L. For
the pGEM standard, use 1 L each of primer and template plus 8 L H2O.
2. Reagents for sequencing reaction (per sample): 2 L 5M Betaine, 1 µL Buffer (400 mM
Tris:HCl, pH 9.0, 10 mM MgCl2) and 2 L ABI BigDye v3.1.
_____µL Betaine _____µL Buffer _____µL BigDye v3.1
Sequencing Date:____________
Number of Sequencing Samples___________
Name of Sequencing File_____________________
Person Performing Analysis___________________
Instrument used for PCR______________________
PCR Conditions: 94°, 60 s; 93°, 30 s55°, 10 s60°, 4 min X 35 cycles; 60° 5 min; 4° hold.
3. Following PCR, either store samples in dark at 4°C or proceed with purification
Samples stored (YES/NO) If stored, date of purification_______________
4. Purification procedure: To each sample, add 10 µL 62.5 mM EDTA (be sure that EDTA is in
the sequencing reaction solution). Next, add 60 L 100% ethanol, mix by inverting several
times and store in dark for 15 minutes. While waiting, set centrifuge temperature to 4°C. Spin
samples at 4000 RPM for 30 minutes-do not exceed or you will precipitate the dye terminators.
Remove liquid from tubes and add 60 L 70% ethanol, invert several times and then spin for 15
minutes at 3000 RPM. Pour off liquid, then invert tubes on top of kimwipe and briefly (~30
seconds) spin at 1500 RPM to remove residual liquid. You may now store the samples in the
freezer protected from light or proceed with reconstitution. Samples stored (YES/NO). Date of
Reconstitution_____________
5. Reconstitution procedure: For running on the 3100 Avant, add 20 µL HiDi formamide and
heat at 95°C for 5 minutes followed by quick chilling at 4°C and then load 10 l onto 96 well
ABI plate in 3100 Avant. Date of Run_______________
Room H260 Health Sciences Building, Box 357610 Seattle, Washington 98195-7610 (206) 616-8718 FAX: (206) 543-3204
edkelly@u.washington.edu http://depts.washington.edu/pceut/pceut_services/index.html