Anyone using "hot start" cycle sequencing?

Darren Stauth stauth at crunch.usgmrl.ksu.edu
Thu Oct 27 11:58:34 EST 1994


In article <gustilo-2610942303220001 at ts7-23.upenn.edu>,
gustilo at pobox.upenn.edu 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 crunch.usgmrl.ksu.edu



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