Sequenase[TM] catalyzed sequencing with dye-labeled terminators

ID: 81

Group:

Methods for DNA sequencing

Prepared by:

Bruce A. Roe (broe@uoknor.edu)

Detail:

Single-stranded dye-terminator reactions required approximately 2 ug of phenol extracted M13-based template DNA. The DNA is denatured and the primer annealed by incubating DNA, primer, and buffer at 65degC. After the reaction cooled to room temperature, alpha-thio-deoxynucleotides, fluorescent-labeled dye-terminators, and diluted Sequenase[TM] DNA polymerase are added and the mixture is incubated at 37degC. The reaction is stopped by adding ammonium acetate and ethanol, and the DNA fragments are precipitated and dried. To aid in the removal of unincorporated dye-terminators, the DNA pellet is rinsed twice with ethanol. The dried sequencing reactions could be stored up to several days at -20degC.

Year prepared: NULL

Date uploaded: 2010-02-26 14:28:18

url:

Laboratory Protocols