PCR: hot start

Hank Seifert h-seifert at nwu.edu
Fri Apr 29 09:26:28 EST 1994


In article <Pine.3.03+.9404281231.A9883-b100000 at fhs.csu.McMas> denomme at FHS.CSU.MCMASTER.CA (Greg Denomme) writes:
>
>
>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
Greg:
We add a homemeade parafin wax bead on the top of the reaction 
without template and enzyme.  Heating to 80C melts the parafin 
and forms a barrier (we have had to adjust the volume of wax in 
the bead to make a minimal complete barrier). The enzyme and 
template are added on top of the wax and amplification done 
under normal conditions.  After the final reaction and cooling, 
we find the wax forming a barrier on top.  I'm not sure why 
this does not work for you.
Hank Seifert
Northwestern University



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