Methodology for sequencing double stranded DNA

Solutions and Buffers for sequencing with T7 DNA Polymerase

NaOH                   Take commercial 1M stock solutions and aliquot them in 
                        1 ml batches.  You must aliquot them because of the 
                        transient neutralization of the NaOH by atmospheric CO2.

HCl Take commercial 1M stock solutions and aliquot them in 1 ml batches.

Annealing Buffer 1M Tris HCl, pH 8, 100 mM MgCl2.

Labelling Mix 10 ÁM flourescin-15-dATP, 1 ÁM each of dCTP, dGTP, dTTP

Extension Buffer 10 ÁM MnCl2, 304 mM NaCitrate, 324 mM DTT

Termination Mixes 1 mM dATP, 1 mM dCTP, 1 mM c7dGTP, 1 mM dTTP, 5 Ám ddNTP, 50 mM NaCl, 40 mM Tris-HCl (pH 7.4)

Stop Mix (Deionize) Mix 50 ml commercial formamide with 5 g Bio-Rad AG 501-X8, 20-50 m on a stirrer for 30 minutes at room temperature. Filtrate through a MN 615 paper filter. Adjust Dextran Blue 2,000,000 concentration to 6 mg/ml and EDTA (pH 8.3) to 20 mM.

Enzyme T7 DNA polymerase may be used from Pharmacia, Promega or USB according to their instructions. Usually the enzymes are purchased together with the appropriate enzyme dilution buffer.

For more information please refer to Sequencing Double Stranded DNA using dye-labelled primer and Sequencing Double Stranded DNA with fl-15-dATP.

Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.

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Updated 1/20/98