PCR

rhubner at molbiol.ox.ac.uk rhubner at molbiol.ox.ac.uk
Thu Apr 21 04:05:25 EST 1994


Hi there,
 your method is NOT a real hot start, because your polymerase is present when
the temperature increases from ice-cold to denaturation temperature! To make a
_hot start_ you must arrange for the polymerase _starting_ to work at above the
extension temperature (i.e. added in middle of "denaturation" step through oil,
or separated from main mix during temperature increase by waxy material, or ?
... find a trigger for polymerases to start only after an initial heating
step?)  
 All the best,
   Roland
+++++++++++
 
In article <Pine.3.89.9404131147.A24938-0100000 at unixg.ubc.ca>, brunstei at UNIXG.UBC.CA (John Brunstein) writes:
> Hello Jiri,
> 	There was a thread a few months back where it was suggested that 
> ordinary petroleum jelly (aka Vaseline) would do quite well (sorry, i 
> don't remember who said they had used this).  However, our lab has a 
> really simple method for getting hot starts, and I was wondering if 
> perhaps there is some glaringly obvious reason why other labs don't do 
> this as well:
> 	we just set up the PCR reaction on ice and KEEP it on ice until 
> the thermocycler blocks hit 90C or so; THEN put the tubes in and go have 
> a coffee.  Not only don't you have to buy gems or squish vaseline into 
> tubes but you can mix the reaction mixture yourself instead of hoping 
> convection will do a good enough job for you after the phases come in 
> contact.  
> 	I would be really interested in hearing other people's opinions 
> on this seemingly simple method.  Is there a reason no-one else does it 
> this way?
> 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.
>> 
>> 



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