bigdye dilutions

Alberto Pallavicini alberto at eos.bio.unipd.it
Wed Sep 23 18:53:08 EST 1998


Hi to all sequencers

I work in a lab involved in many sequencing projects and we have two ABI 377 
96 lanes.
Sometimes we have the "normal" gel problems but I have solved them using your 
suggestions.
When I switched from dRhod to Bigdye mix I optimised a new recipe according to 
those posted here. (1ul mix and sometime less, with supplemented buffer, 3.2 
pmol of primer and 200 ng of plasmid DNA to a final volume of 10 ul) now with 
the new batch of mix arrived on July I hace obtained results that are 
completely unsatisfactory.  

Instead, when I got  the Bigdye mix for the first time I tried to use 4 ul of 
Bigdye mix and load on gel almost all of the reaction after EtOH/NaOAc 
precipitation but the sequence image was so strong that the analysis of the 
signal was impossible.
Now I have retried a 4 ul recipe using the pGEM test DNA and the gel image was 
fainter with well resolved bands.

I am suspecting that the Big dye mix is now already diluted by the producer 
whithout a correspondent reduction in the price.

Did somebody get the same results?
Any other hypothesis for my results?

CIAO

Alberto Pallavicini
CRIBI Biotechnology Centre
Università di Padova





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