Rebalancing Big Dye terminator kits

Phillip San Miguel pmiguel at purdue.edu
Sun Nov 21 17:35:19 EST 1999


    The problem: on the 3700 short products apparently are injected
preferentially over longer products--thus one tends to get much stronger
signal at the beginning of a read than towards the end. Stephen Lasky
mentioned that shortening the sample injection time ameliorates this
problem. I've done this, but still often see signal in the beginnning so
high that it maxes out the sequence analysis software and I lose
sequence towards the end because the signal is too weak.
    I hear from PE-ABI that the "rebalanced" Big Dye kits (that favor
longer products) won't be available until Jan 2000. These probably just
adjust the ratio of deoxys to teminators to favor deoxys. So I could get
the same effect by adding extra nucleotides. PE won't say what
concentrations nucleotides are in the Big Dye kit. Does any one know or
want to take a guess? Most troublesome would be getting the dITP
concentration in the right range, I guess.
    The maddening aspect of PE-Bio's tardiness in releasing this kit is
that at GSAC it was mentioned the the "rebalanced" or "reoptimized" kit
gave better results for the 377 as well.

Phillip San Miguel
Purdue Genomics Center





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