Real time PCR
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Mon Apr 24 05:59:24 EST 2000
In article <#oGC8hbr$GA.78 at net003s>, Jaco Hendriks
<jaco.hendriks at hetnet.nl> writes
>what's the difference between this one and the ones of Perkin Elmer, Roche
>and Bio-Rad? I would be very interested in this answer considering the fact
>we are looking for the best real-time PCR apparatus right now (and off
>course for the money we need to buy one).
>b.hendriks at wkz.azu.nl
It uses standard 0.2ml microtubes not glass so no sharps and no funny
PCR (or worse RT-PCR) buffer chemistry due to the glass. It takes 32
tubes and spins them at 600rpm in basically a 45 degree rotor in heated
air. Fluorescent sampling is done with two LEDs and
photomultipliers/10nm filters. It samples each tube in turn, exciting
horizontally through the side of the tube, picking up the fluorescence
vertically at 90 degrees. It samples each tube in turn whilst spinning
and does this for 60 revolutions measuring every second revolution i.e.
each tube is measured 30 times over a six second period then average
fluorescence is reported. This is slightly different from a Lightcycler
where the 32nd sample is measured 6 secs after the first. Speed of the
machine is identical to a PE9600 i.e. no where near as fast as a
LightCycler. But once again no glass, standard consumables, 20ul
reactions (haven't tried 10ul) and RT-PCR to high sensitivity should be
a doddle, unlike my experience with the LightCycler. Fluorescence is
monitored on two channnels (will be three in due course) i.e. FAM/Sybr
Green and JOE/HEX/VIC/MAX
>From my initial results of playing this weekend and running samples out
on agarose gels, the product yield, lack of false priming products and
replicates are all better on the Rotor-Gene than my 9700 (when running
in 9600 mode). A pleasant surprise as I thought the 9700 was pretty good
anyway. To be better is a bonus. Presumably this is because as the
samples are all in the same air flow and spinning, the temperatures are
identical unlike a 96 well block where there are variations across the
block. The other thing is that the heated air stays in the rotor chamber
and only gets exhausted during cooling whilst the LightCycler passes
hot/cool air continually past the capillaries so heating the lab!
Anyway back to more playing.
PS the Gel Scan system is equally as nice but I've only run it once so
far. For more info. http://www.corbettresearch.com
Just a disclaimer (for the first time ever!). We are not acting as
distributor of these machines. I'm just an end user.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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