perkin elmer vs light cycler

John F Mackay MackayJF at bmnz.co.nz
Tue Aug 31 16:56:35 EST 1999


Hi,

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
protocol.


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.

Cheers,

John
Roche NZ




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