ramp time & cycle seqn

saboteur at BORCIM.WUSTL.EDU saboteur at BORCIM.WUSTL.EDU
Thu Feb 2 12:40:02 EST 1995


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.

I tried the ABI single-tube seqn reactions on a stratagene machine we had
out on a test drive, one of those ones that has 4 blocks and a robotic arm
that physically picks up the tubes from one block and drops them onto another.
Presumably this "eliminates" ramp time. The reactions didn't work. Using the
same melt/anneal/extension times & temps on a PE 9600 we also had out on loan,
they did work, and beautifully. Note this machine still ramps, but is one of
the fastest "rampers" around. My conclusion is that it probably is important
to have *some* ramp time, but you don't need much. We got the PE 9600.

Brett Lindenbach
Lucille P. Markey Student in Human Pathobiology
Program in Immunology
Washington University - St Louis
brett at borcim.wustl.edu





More information about the Methods mailing list