ABI PRISM BigDye SEQ problem!!!

tom chappell t.chappell at ucl.ac.uk
Wed Feb 10 12:19:57 EST 1999


RPrestonic <rprestonic at aol.comUNSPAM> wrote:

> >In fact, I did get a good sequence with these primers when using
> >a dRhodamine Kit from the same manufacturer.
> 
> You did get? When? During same experiment as BigDye? Or last
> month, with a different phase of moon.
> 
> 
> >
> >I cannot see any good reason why BigDye should not-to-allow
> >to get sequence with AmpliTaq DNA polymerase FS and T3 & T7.
> 
> True.  It's a Ready Reaction kit, right? Everything is already in it
> except the primer and template, right? It should work.  Does a parallel
> labeling reaction (same time, same cycler, same 310 run)  with the
> dRhodamine kit also fail?

And real important, make sure the 310 is set up for BigDyes. The colors
are different than the Rhodamine kit--really different. I don't have the
book right in front of me, but even the wavelengths of each of the 4
bases relative to each other are altered in going from rhodamine to
bigdyes. A is longer wavelength that T in one kit, shorter wavelength in
the other, etc.
 




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