Overwrites to biology. The speculations of XIX century by Ritterbush P

By Ritterbush P

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3. Discard the solution, and add 50 µL of HEN buffer containing a 1:10,000 dilution of the SYBR Green I (the iFRET donor) directly from the tube as supplied by the manufacturer (see Note 10). 4. Detection of Melting Curves Using iFRET 1. 3°C per second. 2. Export the data file to a spreadsheet (such as Microsoft Excel®) and extract the appropriate fluorescence and temperature data (see Notes 11 and 12). 3. A plot of the fluorescence vs temperature data produces a characteristic melting curve. When the Tm is reached, the probe and target separate and the FRET reaction stops as marked by a large drop in fluorescence (see Note 13 and Fig.

There are several ways to screen combinations of reporter/quencher pairs. A series of quencher–reporter pairings were recently tested by Marras, Kramer, and Tyagi. They used complementary oligos with 5'-reporters and 3'-quenchers to bring the dyes directly together or at staggered distances to measure, respectively, contact-mediated and FRET quenching efficiencies (5) (Fig. 3). This method of bringing the dyes together in order to measure contact (static) quenching holds the reporter and quencher in a fairly constrained and fixed relative orientation.

The labeled oligonucleotides had to be purified with reverse-phase high-performance liquid chromatography. The detection of hybridization is limited by the concentration of the target RNA molecules and sensitivity of the detector devices; thus, relatively abundant samples can be analyzed using a conventional spectrofluorometer (F-5000, Hitachi), whereas more dilute samples should be measured using a fluorescence microscope equipped with a detector device (such as a C-CCD camera: C4880-40, Hamamatsu Photonics) or a confocal laser-scanning microscope system (µ-radiance, Bio-Rad, Hercules, CA).

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