Spin down first strand reaction, then add 1 ul T7-(dT)24 primer.

Rinse the blocks in dH20 and transfer to a vial such as a scintillation vial.

Oligo(dT) primer generates a high frequency of …

To study objects we must interact with them. Generally, the technique of interaction is determined by the size of the object. Thus, an object of macroscopic size can be studied by direct contact. However, microscopic objects, such as the cell and its organelles, must be studied with agents of similar or smaller size. Cells in are roughly 3–30 microns in diameter, thus, light with wavelength in the visible range (~500 nm) is an ideal interacting agent. The set up of light microscopes affords a resolution that is about half the wavelength of light employed. Thus, light microscopy is useful for examining cells and cellular substructures on the order of 200–300 nm or larger. However, vesicles (often 50 nm in diameter), and other objects of similar or smaller scale cannot be resolved using current setups. Although, in principle, light of very short wavelength (e.g. X-rays) could be used to resolve smaller cellular structures, such light is too energetic, damaging the cell upon contact. In addition, lenses to focus high energy photons do not exist. High resolution can be obtained using electron microscopy. Moving electrons in an electron microscope possess wavelengths on the order of 0.3 nm. Using an electron microscope, the electrons can be used to form resolved images of cellular structures of about 3 nm in size.

Protocol 31: Primary culture of embryonic cells (Kevin Strange and Rebecca Morrison)

Is a random primer or oligo(dT) better for RT-PCR reaction?

Complete RT² miRNA PCR System
Other sources of miRNA reverse transcription assay will not work with the RT² miRNA PCR Arrays and Assays, and cDNA from the RT² miRNA First Strand Kit cannot be used with other source of miRNA qPCR assays. Only the complete RT² miRNA PCR System yields detectable real-time PCR results.

Due to this unique feature, EasyScript Plus™ can synthesize full-length cDNA libraries from RNA templates up to 15 kb in length.

Control:
A built-in miRNA External RNA Control (ERC) helps test for inhibitors of reverse transcription. Control elements on the RT² miRNA PCR Arrays detect template synthesized from the ERC. A reproducible threshold cycle value from this control across your samples indicates a consistently high level of RNA quality and transcription efficiency giving you a greater degree of confidence in your results.


Use for first-strand cDNA synthesis with a reverse transcriptase

1. A Poly-A Polymerase adds a poly-A tail to the miRNA sequence.
2. A primer, containing oligo(dT) and a universal PCR primer sequence, anneals to the poly-adenylated RNA.
3. A Reverse Transcriptase synthesizes and appends to the primer DNA sequence complementary to the miRNA.
4. The final first strand cDNA product contains the miRNA complement sequence, a poly(dT) tract, and the universal PCR primer sequence. It is immediately ready for a real-time PCR assay containing a miRNA-specific and the universal PCR primer.

Oligo(dT) 15 Primer is suitable ..

Visible light can be used to examine , however, in general, bright field and phase-contrast microscopy offers little contrast- making cells and their major components difficult to see. DIC microscopy, however, allows high contrast images to be formed, and is ideal for examining nuclei, nucleoli, and granular structures within cells (; ). In DIC microscopy light passes through a plane polarizer. The resulting polarized light is split into two components using a prism. These components interact with the sample, and are combined above the objective using a second prism. Finally, the transmitted light passes through an analyzer (essentially a polarizer that is polarized at 90 degrees with respect to the original plane of polarization) and on to the observer. The interaction of polarized light with the sample changes its plane of polarization. Thus, only light whose polarization is changed by the sample will be detected by the observer. In this way, highly contrasted images are formed. It should be remembered that shadowing effects seen using DIC microscopy reflect three-dimensional features of the sample. Rather, they reflect the ability of the specimen to interact with polarized light. Current microscopes allow the use of a 100X objective with DIC, giving high magnification and resolution. Some microscopes are equipped with additional lenses, however, these do not afford better resolution.

Concentration; Oligo(dT) 15 Primer

NEB offers several reagents for cDNA Synthesis for use in applications including qPCR and qRT-PCR. For your convenience, reagents are available as kits or standalone products, depending on your needs.