13 Sequencing
195
Procedure
1. In advance, prepare the following on ice:
ln advance
a. dNTPs (labeling mix, 5 x concentrated): Prepare a stock solution (7.5
J.lM each dNTP):
dCTP {10 mM)
dGTP (10 mM)
dTTP (10 mM)
ultrapure water
total volume
store in aliquots at -20°C.
3.8 ~l
3.8 ~l
3.8 ~l
5 ml
5 ml
b. "A, T, G, C" termination mixes: Prepare 4 stock solutions (80 J.lM each
dNTP, 8 J.lM each ddNTP):
.
dCTP (10 mM)
8 ~l
dGTP {10 mM)
8 ~l
dTTP {10 mM)
8 ~l
dATP {10 mM)
8 ~l
ddNTP (10 mM)
0.8 ~l
NaCl (50 mM)
967 ~l
total volume
1 ml
store in aliquots at -20°C.
c. primers: Dilute to a final primer:template molar ratio of about 1:1; start
with 3 J.lglml using a universal primer of 17 nucleotides in length.
d. DNA template: M13 single-stranded template: adjust concentration to
1 J.lg I 7 J.ll, use directly in the sequencing reaction.
Plasmid double-stranded template must be denatured prior to use as
follows:
1. Add 2 J.ll2 M NaOH to 3 J.lg plasmid in 8 J.ll water.
2. Mix, incubate for 10 min at room temperature.
3. Add 3 J.ll3 M NaAc and 7 J.ll water.
4. Add 60 J.ll (=3 vol.) ice-cold ethanol (lOOo/o), mix, place on dry ice I
ethanol for 10 min.