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<h2>bulk measurements</h2>
<h2>bulk measurements</h2>
We planned to measure bulk FRET at the photospectrometer and the RT-PCR. However, the photospectrometer is not sensitive enough to deal with Atto dyes at concentrations below 10 nM (peaks were not visible at all). The RT-PCR was more sensitive, fluorescence signals could be recorded from 50 µl samples with 10 nM Atto dyes.  
We planned to measure bulk FRET at the photospectrometer and the RT-PCR. However, the photospectrometer is not sensitive enough to deal with Atto dyes at concentrations below 10 nM (peaks were not visible at all). The RT-PCR was more sensitive, fluorescence signals could be recorded from 50 µl samples with 10 nM Atto dyes. For first tests, a simple [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Helix_MH_255/256 18 bp DNA double helix] with Atto 550 ddCTP at the one end and Atto 647N ddUTP at the other end was examined. To check the accuracy of the RT-PCR, measurements were set up in duplicate, i.e. 100 µL sample were prepared and subsequently divided into two 50 µl samples, making them exactly identical. However
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FRET

http://openwetware.org/index.php?title=Biomod/2011/TUM/TNT/Results/FRET&action=edit


FRET labels

As FRET labels we use the fluorophores Atto 550 and Atto 647N. The Förster distance for this pair is 6.5nm according to AttoTec. Both dyes are commercially available linked to ddNTPs, so they can be attached to oligonucleotides using terminal transferase. The fluorophores not only exhibit a high stability against photobleaching, but also have excitation and fluorescence spectra that fit to the set-up of the self-made fluorescence microscope in our lab. Thus we have not only the possibility to measure FRET at the photospectrometer and the more sensitive RT-PCR, but can also perform single molecule experiments at our TIRF.

spectrometric characterization of DNA binders

Lots of DNA binders interact with DNA via aromatic systems or do otherwise absorb in the range of UV/VIS light. This could cause background signals, which impair the FRET measurements. Therefore, we recorded spectra of the Atto dyes mixed with the different DNA binders (see Labbook: 11th August and here in Fig. 1).

Fig.1: Excitation and emission spectra of Atto 550 ddCTP resp. Atto 647N dUTP with and without different DNA binders
Atto dyes: 100 nM, DNA binders were added in a concentration according to 10x their KD (see references), i.e.: Hoechst 33258: 22 nM, DAPI: 18 nM, spermine: 48 µM, methyl green: 22 nM, ethidium bromide: 120 µM
EtBr without Atto 550 ddCTP refers to a spectrum with only 120 µM ethidium bromide, while Atto 550 ddCTP corrected for EtBr is the difference spectrum between ethidium bromide and EtBr without Atto 550 ddCTP
buffer: 0.5x TBE, 11 mM MgCl2; slit width: 5 nm; room temperature

Most DNA binders do not exhibit significant effects on the absorption and emission spectra of the Atto dyes and thus can be used for FRET measurements. Methyl green causes major effects and therefore will not be considered further for these experiments. Ethidium bromide absorbs and emits at similar wavelength as the both Atto dyes, but as the difference spectrum proves, the Atto dyes itself are not affected. Consequently, ethidium bromide causes background signals, that must be corrected for, but otherwise can be used in our measurements.
At first, we concentrated on three DNA binding compounds, ethidium bromide as example for an intercalator, DAPI as an minor groove binder and spermine as an major groove binder.


bulk measurements

We planned to measure bulk FRET at the photospectrometer and the RT-PCR. However, the photospectrometer is not sensitive enough to deal with Atto dyes at concentrations below 10 nM (peaks were not visible at all). The RT-PCR was more sensitive, fluorescence signals could be recorded from 50 µl samples with 10 nM Atto dyes. For first tests, a simple 18 bp DNA double helix with Atto 550 ddCTP at the one end and Atto 647N ddUTP at the other end was examined. To check the accuracy of the RT-PCR, measurements were set up in duplicate, i.e. 100 µL sample were prepared and subsequently divided into two 50 µl samples, making them exactly identical. However

Inhalt:

-Results- ggf: FRET und TIRF zusammen auf einer seite. Wichtige Ergebniss graphisch darstellen.

-Discussion- Disskusion der Ergebnisse

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