Sequencing PCR-amplified human hprt cDNA with 373 Stretch

G. Dorado .bb1dopeg at lucano.uco.es
Sun Dec 11 05:41:02 EST 1994


We are experiencing problems sequencing the PCR-amplified human hprt
cDNA (no problem with other PCR sequences, though!) using the 373
automatic sequencer from Applied Biosystems /Perkin Elmer. Typically,
we get too many "N" both using dsDNA or ssDNA from hprt (obtained using
biotinilated primers and Dynal magnetic separation). Reactions were
performed using the Taq cycle sequencing kits of AB/PE. Curiously,
sequencing is OK with classical radioactivity chemistry (using
Sequenase).

We have recently upgraded to the 373 Stretch automatic sequencer
(Applied Biosystems/Perkin Elmer). We wonder if the new Sequenase kits
(ss or ds terminator chemistries) could be worth trying.

Any comments will be most appreciated.

Thanks,

G. Dorado
InternetEmail: bb1dopeg at lucano.uco.es



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