Anyone using "hot start" cycle sequencing?

Darren Stauth stauth at
Thu Oct 27 11:58:34 EST 1994

In article <gustilo-2610942303220001 at>,
gustilo at wrote:

> greetings,
> I was using the Promega fmol cycle sequencing kit and I found that I was
> having some trouble getting all the reagents into the tubes in a timely
> manner.
> It occured to me to use wax to separate the stop solution and primer from
> the rest of the cycle sequence reagents. Has anyone out there tried this?
> My only concern at the moment is that one is working with such small
> volumes (6 ul) that there could be a problem with inadequate mixing of the
> solutions.
> I have performed "hot start" PCRs using a 288 fold degenerate 5' primer
> and a non-degenerate 3' primer and I have gotten good results with it. 1/4
> of a Paraplast bead works great BTW

I'm not quite sure what your trying to explain here.  I perform fmol cycle
sequencing quite often and have always had good results.  I follow the
protocol almost exactly:  mix labeled primer,template,buffer,water-->add
Taq Pol (on ice)-->pipette to mix and add 4ul to each d/ddNTP tube(as long
as the 2ul of d/ddNTP is at the bottom of the tube, the two solutions will
be adequately mixed)-->start PCR program:preheat to 95degrees-->add tubes
to preheated block and start cycling.  The last step in my program is
4degrees indefinitely.  I have left the reactions for 4 hours or more at
this temperature before adding the stop solution and haven't had any
problems.  Also I am never in any rush to do any steps except getting all
the tubes (usually 40) into the preheated PCR machine.  Feel free to email
me if you have any questions 
Darren M. Stauth -- stauth at

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