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, it should be noted that the filters do not excite the fluorescent dyes and measure fluorescence at wavelenghts that are optimal for Atto 550 and Atto 647N.(see above) | 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, it should be noted that the filters do not excite the fluorescent dyes and measure fluorescence at wavelenghts that are optimal for Atto 550 and Atto 647N.(see above) | ||
[[Image: | [[Image:FRET efficiency Spermine skal.PNG]] <br> | ||
[[Image:FRET_efficiency_EtBr.PNG]] | [[Image:FRET_efficiency_EtBr.PNG]] | ||
Revision as of 06:27, 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, it should be noted that the filters do not excite the fluorescent dyes and measure fluorescence at wavelenghts that are optimal for Atto 550 and Atto 647N.(see above)
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