Biomod/2011/TUM/TNT/LabbookA/2011/09/01: Difference between revisions
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The following graphs show no significant correlation between neither spermine nor ethidium bromide concentration and FRET efficiency in this experiment. A concentration of labeled oligos of 10 nM seems to be below the detection limit of the real time PCR gadget. 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 graphs show no significant correlation between neither spermine nor ethidium bromide concentration and FRET efficiency in this experiment. A concentration of labeled oligos of 10 nM seems to be below the detection limit of the real time PCR gadget. 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). | ||
<br> | <br> | ||
<center>[[Image:FRET efficiency Spermine skal.PNG|x220px]][[Image:FRET_efficiency_EtBr.PNG| | <center>[[Image:FRET efficiency Spermine skal.PNG|x220px]][[Image:FRET_efficiency_EtBr.PNG|x220px]]</center> | ||
</div> | </div> |
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1st Sep 2011Real time PCR with MH_255_647N and MH_256_550 adding ethidium bromide and spermine
-> dilution 1:100 in buffer is used for real time PCR Table 1: Pipeting scheme spermine
Table 2: Pipeting scheme ethidium bromide.
Table 3: Pipeting scheme ethidium bromide control without labels: Error: there was added 1 µL of buffer too much.
Atto 550 was excited at 545 nm and fluorescence detected at 568 nm, Atto 647N was excited at 635 nm and detected 665 nm, thus matching the conditions of the real time PCR's optical filters. ResultsThe following graphs show no significant correlation between neither spermine nor ethidium bromide concentration and FRET efficiency in this experiment. A concentration of labeled oligos of 10 nM seems to be below the detection limit of the real time PCR gadget. 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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