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

josmarlangner at yahoo.de josmarlangner at yahoo.de
Thu May 26 09:25:58 EST 2005


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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