Big Dye Sequencing on 377

Phillip San Miguel pmiguel at purdue.edu
Tue Sep 5 05:12:29 EST 2000


Andy Vierstraete wrote:

> [...]>Frazer Murray wrote:
> >
> >> Hi everyone
> >> has anyone else been having trouble with their sequencing reactions
> >> lately? I've found I've had to increase the amount of sequencing reagent
> >> from 2ul to 3ul (in 10ul total volume) which has considerably increased
> >> costs. I've started on a new library but the old one was showing the same
> >> problem so I don't think that was the cause.
> >> It would be interesting to hear if others had noticed this as well.
> >> [...]
>
> I have noticed the same problem : we used 4 times diluted mix, and now I can
> only dilute it 3 times, or the signal is to weak.  Maybe PE made the solutions
> less concentrated ???
> [...]

What version of the big dye terminator kit are you using? If you have switched to
version 2.0, then your concentration of terminators is lower. V. 2.0 is meant to
give longer average sequence product length by reducing the amount of terminators.
The figure I've heard is that there are 1/2 the concentration of terminators in v.
2.0 compared to the original big dye kit.

Phillip San Miguel
Purdue Genomics Core Facility




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