198
KLAUS DEICHMANN
Procedure
ln advance 1. In advance, prepare the following on ice:
a. dNTPs: Prepare a stock solution (150 f.lM each dNTP):
dCTP (10 mM)
15 !Ü
dGTP (10 mM)
15 ,ll
dTTP (10 mM)
15 J.ll
dATP (10 mM)
15 J.ll
ultrapure water
940 J.ll
total volume
1000 J.ll
store in aliquots at -20°C.
Note: Sequencing with Taq polymerase is suited for use of diTP or 7-deazadGTP to avoid compression bands. In this case 75 f.ll (750 ~-tmol) diTP (10
mM) is substituted for dGTP.
b. Reaction mix: Prepare a stock solution. The total volume should be
n+ 1 times the volume for one reaction (38 ~-tl):
5 x TACS buffer
dNTP mix
DyeDeoxy™ A-Terminator
DyeDeoxy™ C-Terminator
DyeDeoxy™ G-Terminator
DyeDeoxy™ T-Terminator
AmpliTaq™ (8 U/J..tl)
can be kept at 4°C for up to a month.
8 vol.
2 vol.
2 vol.
2 vol.
2 vol.
2 vol.
1 vol.
c. Primers: Dilute to a final molar ratio of about 3:1 to 4:1; start with 5 f.l,g/
ml using a universal primer of 17 nucleotides in length.
d. DNA template: Adjust concentration:
- M13 single-strand template: 500 ng/f.l,l
- Plasmid double-strand template: 1 f.l,g/f.l,l