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

cot assay (DNA-reassociation kinetics) on realtime pcr machine - Protocols and Methods Forum

cot assay (DNA-reassociation kinetics) on realtime pcr machine - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 05-26-2005, 08:50 AM
josmarlangner@yahoo.de
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Default cot assay (DNA-reassociation kinetics) on realtime pcr machine



Dear all,
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!!!
Cheers
Josmar

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  #2  
Old 05-26-2005, 09:03 AM
Duncan Clark
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Default cot assay (DNA-reassociation kinetics) on realtime pcr machine

Historians believe that in newspost
<1117097410.406687.236620@z14g2000cwz.googlegroups .com> on Thu, 26 May
2005, [Only registered users see links. ] penned the following literary masterpiece:

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.

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

Duncan Clark
GeneSys Ltd.
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  #3  
Old 05-26-2005, 02:25 PM
josmarlangner@yahoo.de
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Default cot assay (DNA-reassociation kinetics) on realtime pcr machine

Dear Duncan,
I have done initial runs, basically nothing more than up and down in
temperature, one DNA concentration (~30 nM) titrated against 3 SGI
concentrations (10x, 1x, 0,1x) in PCR buffer, nothing else. The ABI
7900HT with SDS 2.1 software can be set to do low ramp rates be it up
or down. The software however cannot analyze such a run. So I will have
to extract the raw data from the result file and process them in excel
to plot emitted light over temp. I would like to set up an assay to
monitor the loss of complexity of selex libraries over the rounds. The
less complex, the faster the reassociation kinetics. To start with, I
would be glad to see a difference between a naive library with every
molecule unique and a corresponding molecule with a defined sequence in
place of the random region.
Cheers,
Josmar

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assay , cot , dnareassociation , kinetics , machine , pcr , realtime


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