-dilution of "Big Dyes"

Phillip San Miguel pmiguel at purdue.edu
Thu Dec 21 07:52:54 EST 2000


"Woodland, M P" wrote:

> [...]What  is  different  about  capillary  based  systems  that  dillution  and
> v2.0  apparently  gives such  poor  results?
> [...]

    I didn't mean to suggest that v. 2.0 gave poor results in my hands. I haven't tried it. But my
understanding is that Applied Biosystems reduced the concentration of terminators in v. 2.0 to
produce more signal farther from the primer and less signal near the primer. That is, they modified
the ddNTP/dNTP ratio to favor longer products by decreasing the ddNTP concentration. Hence I expect
less overall signal since the ddNTP's carry the flourescent tag in terminator sequencing. With full
or 1/2 reactions I don't expect any problems--there is plenty of label to go around (unless one is
sequencing BAC or bacterial DNA?). But at 1/8th or 1/16th reactions I expect trouble. But I may be
wrong...

Phillip San Miguel
Purdue University Genomics Core Facility



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