Dynabeads Oligo(dT)25 can be applied directly to PCR (or any other enzymatic reactions) as the beads do not inhibit or reduce enzymatic activity. This is because the iron is safely sealed off inside the Dynabeads by an inert polymer shell.
Adding the Dynabeads-mRNA complex directly to the RT-PCR offers benefits: Elution, and thereby dilution, of the mRNA is avoided. With mRNA hybridized to the beads, the bead-bound oligo-dT act as primers for first strand cDNA synthesis, producing a bead-bound cDNA library. Construction of such a solid-phase cDNA library is of special benefit when mRNA is isolated from very small samples. Solid-phase cDNA libraries have been generated from a single cell using this technology. In addition, the solid-phase cDNA library can be regenerated and reused again and again, for almost unlimited number of times.
Dynabeads have no quench effect on the fluorescence signal in real-time PCR, but do exhibit autofluorescence which may in some degree affect the intensity of the signal. If observed, this can be compensated for by installing a Dynabeads & water background in the PCR instrument. This background signal intensity can be subtracted from the sample signal intensity in all subsequent real-time PCR experiments containing beads. In this way, Dynabeads can be applied directly in Real-time PCR instruments such as ABI Prism 7000 and 7700 Sequence Detection System (Applied Biosystems) - without interfering with the results of the analysis. Please note that Dynabeads Oligo(dT)25 are not compatible with capillary Real-time PCR machines, e.g. like Roche LightCycler™ instrument.
This video shows the 3 simple protocol steps + some tips & tricks:
- Bead preparations & protocol scaling
- Ab choice (type &concentration)
- How to use the magnet
- Pipetting & mixing
- Crosslinking (optional)
- Direct vs. indirect binding
For Research Use Only. Not for use in diagnostic procedures.