Dye-Deoxy Cycle Sequencing-ramp time?

John H McDonald mcdonald at strauss.udel.edu
Mon Jan 30 18:02:56 EST 1995


In article <Pine.OSF.3.91a.950127195746.12261B-100000 at saul5.u.washington.edu>,
James Gray  <jp at u.washington.edu> wrote:
>	Does anyone know how important ramp time REALLY is in Taq 
>Dye-Deoxy cycle sequencing?  I am using an MJ Research Minicycler using 
>the 0.2ml tubes and the default ramp times on the cycler.  This machine 
>has a noticeably faster ramp time than the recommended Perkin-Elmer 
>machine (1C/sec).  I find that my reads fall off at about 90 bases and 
>the background is high.
>

We do dye-terminator cycle sequencing for the ABI 373 using an Idaho 
Technologies thermal cycler, which has much faster ramp times than your 
poky ol' MJ, and it works fine.  Make sure your extension time is at 
least three minutes, since the modified nucleotides used in 
dye-terminator sequencing get added quite slowly.  Also, try to quantify 
your template; noisy sequence that starts strong, then drops off rapidly 
can be caused by too much template DNA.  

John H. McDonald
Department of Biology
University of Delaware




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