Protocol for reducing BigDye to 1/8

Akira Tanigami atanigam at otsuka.genome.ad.jp
Sat May 30 08:39:12 EST 1998

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,

                        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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