injection solution which reduces rfu

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injection solution which reduces rfu

dna0001


Hi all
    I work in a core lab setting, and have recently upgraded my Applied
Biosystems 3100 Genetic Analyser to a 3130xl running POP-7. A client wants to
perform fragment analysis with multiplexed PCR products, but often the peaks of
interest are off-scale (i.e. > 7000 rfu). To the best of my knowledge, Gene
Mapper v4.0 and Peak Scanner v1.0 will not analyse these samples due to the
off-scale peaks. I've tried adding 0.1mM EDTA to the samples (we generally use
HiDi formamide only for injection), but have found that this can either
increase or decrease signal strength. Can anyone suggest a solution which will
decrease signal strength? Will low melting point agarose achieve this? Any
suggestions would be appreciated.

Many thanks

Oliver

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Re: injection solution which reduces rfu

Phillip SanMiguel
[hidden email] wrote:

>
> Hi all
>     I work in a core lab setting, and have recently upgraded my Applied
> Biosystems 3100 Genetic Analyser to a 3130xl running POP-7. A client wants to
> perform fragment analysis with multiplexed PCR products, but often the peaks of
> interest are off-scale (i.e. > 7000 rfu). To the best of my knowledge, Gene
> Mapper v4.0 and Peak Scanner v1.0 will not analyse these samples due to the
> off-scale peaks. I've tried adding 0.1mM EDTA to the samples (we generally use
> HiDi formamide only for injection), but have found that this can either
> increase or decrease signal strength. Can anyone suggest a solution which will
> decrease signal strength? Will low melting point agarose achieve this? Any
> suggestions would be appreciated.
>
> Many thanks
>
> Oliver
>
Hi Oliver,
You could try reducing the injection time. Or ask your customer to load
less sample.

Seems like adding salt (like EDTA) just competes with product such that
larger fragments diminish in intensity, but smaller ones stay about the
same.
Phillip

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