Sheila E. Norton

March 14, 2000

Automated DNA Sequencing was conducted at the UNMC Eppley Cancer Center Molecular Biology Core Facility at the University of Nebraska Medical Center, Omaha, NE. Cycle sequencing was performed using ABI Prism BigDye Terminator Ready Reaction Kits containing AmpliTaq® DNA Polymerase (Part No. 4303153, PE Biosystems, Foster City, CA). The Ready Reaction solution was diluted 1:1 with 5X ammonium sulfate buffer (ASB) to make "half reactions". 10X ASB contained 200 mM Tris-HCl, pH 8.8, 15 mM MgCl2, 100 mM KCl. To each half reaction (8ul) was added 4 picomoles of primer (4 ul) and a sufficient amount of purified plasmid or PCR products (8ul). The amount of DNA template used in the sequencing reaction varied according to the size (i.e., kilobases) of the entire DNA template. Typically, 100 nanograms is used for every kilobase of DNA template. 5% DMSO is added as needed. The constituted reactions were denatured at 95^oC for 5 minutes. Cycling began with denaturing at 95^oC for 15 seconds, annealing at 55^oC for 15 seconds and extension for 3 minutes at 60^oC and the cycle repeated for a total of 100 total cycles in a Biometra Model Uno II Thermocycler (Biometra, Inc.,Tampa, FL). The reactions were then purified on G-25 Sephedex resin by centrifugation to remove unreacted fluorescent BigDye Terminators. The purified reactions were dried on a vacuum centrifuge and reconstituted in formamide/Tris-EDTA (4:1) sample buffer, denatured for 3 minutes and snap-cooled on ice. Samples were then loaded onto a 48 cm well-to-read sequencing gel made from Page Plus 40% Concentrate (Amresco, Solon, OH) to a final concentration of 5% acrylamide and 6M urea. Electropheoresis was carried out for 10 hours at 51^oC on ABI Prism Model 377 Automated DNA Sequencer (PE Biosystems, Foster City, CA) equipped with a Macintosh G3 Power PC computer with ABI Prism 377-96 Collection Software (version 2.5) and Sequencing Analysis Software (version 3.3).

We appreciate acknowledgement of the UNMC Eppley Molecular Biology Core Facility in presentations and manuscripts. We are supported by the following grant: