cot assay (DNA-reassociation kinetics) on realtime pcr machine

Duncan Clark blackhole at
Thu May 26 04:03:32 EST 2005

Historians believe that in newspost 
<1117097410.406687.236620 at> on Thu, 26 May 
2005, josmarlangner at penned the following literary masterpiece:
>has anyone ever performed a cot assay for DNA-reassociation kinetics on
>a realtime pcr machine? Plotting SGI emission over temperature during a
>low ramp cooling down phase, let's say from 95° to 50° should be all
>I need. How much DNA is needed for a good signal? How much SGI is
>needed (Sigma does not provide the concentration of the dye, they just
>say it's 10000x). Any hints are appreciated!!!

The real problem may be that most real-time machines ramp UP in 
temperature for the dissociation curves, not down. I know I can't alter 
three different instruments I have here to ramp down, however it may be 
possible on the LightCycler. I have one also, but haven't checked. The 
LC is also probably one of the very few which you can alter the ramp 
rate with as well.

A SYBR Green I level of 0.25x should be fine.

I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.

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