13 Sequencing
197
Subprotocol 2
Standard Protocol for Sequencing with Taq Polymerase
In the following, a standard protocol for sequencing with the chain termination method using Taq polymerase (AmpliTaq™, Perkin-Eimer Cetus)
and dye-terminators is given. Taq polymerase allows linear amplification of
extension products using a thermal cycler. Furthermore, its heat-resistance
allows the direct use of double-stranded DNA without prior denaturation.
Annealing, labeling and termination are performed in one tube simply by
altering the temperature.
Materials
• DNA template (single-stranded or double-stranded DNA)
• Universal oligonucleotide primer( -s)
• Thermus aquaticus polymerase ("Taq") (e.g. AmpliTaq™, Perkin-Eimer
Cetus)
• dNTPs(dATP,dCTP,dGTP,dTTP)
• Dye-ddNTPs (e.g. DyeDeoxy™ Terminators, Perkin-Eimer Cetus)
• Stop solution (5 vol. deionized formamideplus one vol. 50 mM EDTA
(pH 8.0))
• Ultrapure H20
• Mineral oil
• Phenol:H 20:Chloroform (68:18:14 v/v)
• 2 M sodium acetate, pH 4.5
• Ethanol 70% and 100%
• 5 x TACs buffer (400 mM Tris-HCl, 10 mM MgClz, 100 mM (NH4)zS0 4,
pH adjusted to 9.0)
Reagents
Solutions
• Thermal cycler ( e.g. Perkin Eimer Cetus Model 480, Biomed Thermal Supplies
Cycler 60)
• 0.6 ml microcentrifuge tubes
• 1.7 ml microcentrifuge tubes
• Eppendorf microcentrifuge