PCR

Stephen Scharf sjscharf at netcom.com
Thu Apr 14 11:39:04 EST 1994


In article <Pine.3.89.9404131147.A24938-0100000 at unixg.ubc.ca>,
brunstei at UNIXG.UBC.CA (John Brunstein) wrote:

> 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?
Actually, I do a very similar technique: I make up my master mix w/o
enzyme, and  prepare the DNA samples, and then put both on ice. I set the
TC to hold at 80 deg. C., linked to the PCR cycling file. I then add enzyme
to the master mix, and keeping it on ice, I quickly add this the sample
tubes, which then go directly into the TC, and add oil after every 8
samples or so with a repeating pipettor.  As soon as the samples all have
the mix added, I start the PCR program. I chose 80 deg. C. as I figured it
would be too high for primer dimer to form, and to get any significant
extension, but wouldn't kill enzyme as the tubes are sitting in the TC
waiting to go. I've dubbed this technique "poor man's hot start". Seems to
work really well. 
-- 
Stephen Scharf
sjscharf at netcom.com
CompuServe 72070,750



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