"perkin elmer vs light cycler"
WeininS at prose.dpi.qld.gov.au
Fri Sep 10 01:55:20 EST 1999
The message from Roche is terribly rude, especially when it's from the
supplier to this discussion group.
BSA is, or was, the most important ingredient in the PCR on a capillary
machine (including light cycler)as specified by the manufacturer long before
the involvement of Boehringer. Who cares about Roche?
Can the people from Roche at least do some home work with your own(?)
products and their history before you open your big mouth here.
Leslie Research Centre
13 Holberton Street
PO Box 2282,
Toowoomba, QLD 4350
Email: weinins at dpi.qld.gov.au
From: MackayJF at bmnz.co.nz <mailto:MackayJF at bmnz.co.nz> ("John F Mackay")
Subject: Re: perkin elmer vs light cycler
Date: 31 Aug 1999 14:56:35 -0700
Like Charles in a previous post, I also have a commercial association with
the Roche LightCycler but in my role, have adapted nearly a dozen block
protocols to the LightCycler. With one exception (a crudely-purified
parrafin section) we have hit it first time up.
I do not think there are any special tricks however here are some points:
- yes, we use the kits. One, they're optimised for the capillary reaction
and two, minimal pipetting with master mix (easy source of errors). And I
work at Roche so go figure! (Who was talking about BSA??!!)
- I use about 0.5mM more MgCl2 (final conc.) than used in the block
eg. block protocol for 250bp product using 1.5mM MgCl2
95 degrees 5 minutes
95 degrees 30 seconds}
55 degrees 30 seconds} x 30
72 degrees 30 seconds}
72 degrees 5 minutes
On the LightCycler, using 2mM MgCl2, this might be:
95 degrees 2 minutes
95 degrees 0 seconds }.......................standard
55 degrees 10 seconds} x 40
72 degrees 10 seconds}.......................product length in bp /25
Melting curve if required and cooling step
I have run programs with similar numbers of cycles on both 2400 and the
LightCycler and run both sets of products on a gel. There were very sharp
bands with the LightCycler products and this product 'definition' has been
documented by Idaho with their RapidCycler.
There is an adaptation guide available - hit up your Roche rep or account
manager for one.
More information about the Methods