PCR: hot start

[Greg Denomme]

On 27 Apr 1994, Hank Seifert wrote:

> [stuff deleted]
> 
> >> > On Wed, 13 Apr 1994, Mgr.Jiri Drabek wrote:
> >> >
> >> >> Hello, netters!
> >> >> Please, does anybody know alternative (cheap, home made) for PERKIN-ELMER
> >> >> AmpliWax PCR gem (used for hot start)?
> >> >> Any answer will be appreciated.
> 
> We and others have published that ordinary Parafin Wax works 
> fine for hotstart.  {Wainwright & Seifert, 1993.  BioTechniques 
> 14:34-36, and a Trends in Genetics paper in 1993}. In fact, I think 
> AmpliWax is just parafin.
> 

I have used ordinary Parafin and Histology grade wax and have not gotten
them to work like the Ampliwax gems :(   For the Ampliwax protocol, there
is a lower PCR mixture, then the wax (melted and re-solidified) then an upper
PCR mixture.  After the hot start, the wax 'inverts' to the top.  Parafin Wax
and the Hstology grade wax do not do this.
                              ^^^
Am I missing something?  If all you want is to melt wax ON TOP of a PCR
reaction mixture then sure any wax (or 'ampligrease') will do.  When
starting with an RT-PCR ssDNA then the DNA and primers should be kept
separate until the temperature is above the annealing temperature.  This
is a true 'hot start'.

If anyone has a wax that 'inverts' like Ampliwax or know to get the wax to
invert, please post.

Cheers,
Greg
denomme at fhs.csu.mcmaster.ca