Biomod/2011/TUM/TNT/LabbookA/2011/09/01: Difference between revisions
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The following links show that no significant connection between spermine or Ethidium Bromide concentration and FRET efficiency could be detected in this experiment. A concentration of labeled oligos of 10 nM seems to be below detection limit of the Real Time PCR machine. Additionally, the machine has only filters at x nm which is not the optimal wavelenght for the measurment of the fluorescent dyes Atto 550 and Atto 647N. | The following links show that no significant connection between spermine or Ethidium Bromide concentration and FRET efficiency could be detected in this experiment. A concentration of labeled oligos of 10 nM seems to be below detection limit of the Real Time PCR machine. Additionally, the machine has only filters at x nm which is not the optimal wavelenght for the measurment of the fluorescent dyes Atto 550 and Atto 647N. | ||
http://openwetware.org/wiki/Image:FRET_efficiency_Spermine.PNG | http://openwetware.org/wiki/Image:FRET_efficiency_Spermine.PNG <br> | ||
http://openwetware.org/wiki/Image:FRET_efficiency_EtBr.PNG | http://openwetware.org/wiki/Image:FRET_efficiency_EtBr.PNG | ||
Revision as of 02:31, 12 September 2011
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Real time PCR with MH255_647 and MH256_550 adding ErBr and Spermine
-> dilution 1:10 in buffer is used for RtPCR
-> dilution 1:100 in buffer is used for RtPCR Pipeting scheme spermine:
Pipeting scheme spermine control without labels: Error: there was added 1 µL of buffer too much
Pipeting scheme ethidium bromide control without labels: Error: there was added 1 µL of buffer too much
Atto 550 was excited at 545nm and fluorescence detected at 568nm, Atto 647N was excited at 635nm and detected 665nm, thus matching the conditions of the RT-PCRs optical filters.
ResultsThe following links show that no significant connection between spermine or Ethidium Bromide concentration and FRET efficiency could be detected in this experiment. A concentration of labeled oligos of 10 nM seems to be below detection limit of the Real Time PCR machine. Additionally, the machine has only filters at x nm which is not the optimal wavelenght for the measurment of the fluorescent dyes Atto 550 and Atto 647N. http://openwetware.org/wiki/Image:FRET_efficiency_Spermine.PNG
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