194
KLAUS DEICHMANN
suppliers offer sequencing kits with standard protocols. It is always advisable to start with the standard protocols, optimizing reaction conditions as
experience accumulates.
Subprotocol 1
Standard protocol for T7 DNA polymerase (Sequenase™)
Materials
Reagents • DNA template (single-stranded or double-stranded DNA)
• universal oligonucleotide primer( -s)
• modified T7 DNA polymerase (e.g. Sequenase™, USB)
• dNTPs(dATP,dCTP,dGTP,dTTP)
• ddNTPs(ddATP,ddCTP,ddGTP,ddTTP)
• 5 xreaction annealing buffer (Tris-HC1200 mM (pH7.5), MgClz 100 mM,
NaCl250 mM)
• stop solution (95o/o Formamide, 20 mM EDTA, 0.05% Bromophenol Blue,
0.05% Xylene Cyanol FF)
• a[ 35 S]dATP (1000 Ci/mmol)
Solutions • Ultrapure H20
• Dithiothreitol (DTT) 100 mM
• 2M NaOH
• 3M sodium acetate, pH 4.5
• Ethanol 70o/o and 100o/o
• 10 xTBE buffer (121 g Tris base, 7.4 g EDTA, 53.4 g Boric acid in 1000 ml
A.D., pH adjusted to 8.3)
• P/CIA = salt saturated phenol:chloroform:isoamyl alcohol (25:24:1 v/v)
Supplies • water bath at 37°C
• 1.7 ml microcentrifuge tubes
• microcentrifuge
• Ethanol I dry ice bath
KLAUS DEICHMANN
suppliers offer sequencing kits with standard protocols. It is always advisable to start with the standard protocols, optimizing reaction conditions as
experience accumulates.
Subprotocol 1
Standard protocol for T7 DNA polymerase (Sequenase™)
Materials
Reagents • DNA template (single-stranded or double-stranded DNA)
• universal oligonucleotide primer( -s)
• modified T7 DNA polymerase (e.g. Sequenase™, USB)
• dNTPs(dATP,dCTP,dGTP,dTTP)
• ddNTPs(ddATP,ddCTP,ddGTP,ddTTP)
• 5 xreaction annealing buffer (Tris-HC1200 mM (pH7.5), MgClz 100 mM,
NaCl250 mM)
• stop solution (95o/o Formamide, 20 mM EDTA, 0.05% Bromophenol Blue,
0.05% Xylene Cyanol FF)
• a[ 35 S]dATP (1000 Ci/mmol)
Solutions • Ultrapure H20
• Dithiothreitol (DTT) 100 mM
• 2M NaOH
• 3M sodium acetate, pH 4.5
• Ethanol 70o/o and 100o/o
• 10 xTBE buffer (121 g Tris base, 7.4 g EDTA, 53.4 g Boric acid in 1000 ml
A.D., pH adjusted to 8.3)
• P/CIA = salt saturated phenol:chloroform:isoamyl alcohol (25:24:1 v/v)
Supplies • water bath at 37°C
• 1.7 ml microcentrifuge tubes
• microcentrifuge
• Ethanol I dry ice bath