Dear Dr. Venatesh,
First, I made a mistake. We use 1/8 of Big Dye (not 1/10). Here
is our protocol for reducing Dye Terminator.
Common protocol Modified Protocol (ul)
Dye Terminator 8.0 1.0
primer 1.0 1.0
10x PCR buffer - 1.75
(Supplied with common Taq polymerase)
template + H2O 11.0 16.25
--------------------------------------------------------
Total 20.0 20.0
Some researchers prefer to use 10x Custom buffer (containing 200mM
Tris-HCl pH 9.0, 25mM MgCl2) instead of PCR buffer to adjust pH.
Cycle sequencing condition in ABI's protocol works well, but we
change it a little bit. Annealing time is 20-30 sec instead of 5 sec. Maybe
as you know, it is helpful to increase annealing time to make more products.
Also we reduce extension time to 3 min from 4 min to decrease total time.
Best Regards,
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Akira Tanigami, M.D., Ph.D.
Otsuka GEN Research Institute
463-10, Kagasuno, Kawauchi-cho
Tokushima, 771-0192, JAPAN
tel. +81-886-65-2888
fax. +81-886-37-1740
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