[Automated-sequencing] Re: injection solution which reduces rfu

Phillip San Miguel via autoseq%40net.bio.net (by pmiguel from purdue.edu)
Wed May 20 06:06:41 EST 2009


dna0001 from flinders.edu.au 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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