John M. S. Bartlett.pdf - Bio-Nica.info

Thermal Asymmetric PCR 357
Fig. 2. Autoradiograph of a PCR product sequenced by thermal asymmetric PCR.
2.2. Purification and Sequencing of the PCR Product
2.2.1. Sequencing Reagents
1. Annealing buffer (5× concentrate): 200 mM Tris-HCl, pH 7.5, 100 mM MgCl 2 , 250 mM
NaCl, 0.1 M dithiothreitol (DTT).
2. Labeling nucleotide mixture (one for each dideoxy nucleotide): Each mixture contains
80 µM dGTP, 80 µM dATP, 80 µM dTTP, 80 µM dCTP, and 50 mM NaCl. In addition,
the “G” mixture contains 8 µM dideoxy-dGTP; the “A” mixture, 8 µM ddATP; the “T,”
8 µM ddTTP; and the “C” 8 µM ddCTP.
3. Stop solution: 95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05%
xylene cyanol FF.
4. Labeled dATP is [α- 35 S] dATP from Amersham, and specific activity should be 1000
to 1500 Ci/mol.
2.2.2. Purification
Purification should be performed in Centricon 30 column (Amicon).
3. Methods
3.1. PCR Conditions
PCR conditions should be as follows: in a total volume of 25 µL, 20 pmol of each
primer, 1× Taq polymerase buffer, and 1 U of Taq polymerase were incubated with 50 ng