Protocol for reducing BigDye to 1/8

Akira Tanigami atanigam at otsuka.genome.ad.jp
Mon Jun 1 14:30:19 EST 1998


Dear Dr. Venatesh,

> Can you let me know conc of template and primer you use in your
> reaction ?

	We use it as ABI recommends. For PCR products, we use 2-3 ul of
PCR. I usually purify PCR product by TAKARA Suprec-02 (Millipore makes the
same product). For plasmid, 0.1 - 1.0 ml culture of bacteria (Depends on the
vector). Maybe 100 - 500 ng. For BAC, I use 2.0 ug of DNA. But, some
researchers tell 0.4 ug is enough when BAC DNA is sonicated. When we tried
it, we could not get good chromatogram. So I'm not sure and I want to know
about it.

	For primer, 2 - 100 pmol. But, someone strongly recommend too much
primer makes sequence-reading short.



                                                        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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