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second strand cDNA synth without primer? - Molecular …

Add 2 μl T4 DNA polymerase (NEB, 3 unit/μl) and continue the incubation at 16°C for 30 minutes. Add 80 μl phenol/chloroform (25:24) mix, vortex, and spin at 14,000 rpm in an Eppendorf centrifuge at room temperature for 3 minutes. Transfer the supernatant to a new tube. Repeat extraction once. Recover the double-stranded cDNA from reaction solution, using 10 ng DstNI digested pBR322 as carrier, by GENECLEAN II (BIO 101) according to manufacturer's instruction. Elute cDNA into 29 μl Tris (pH 8.0).

Second-Strand cDNA Synthesis Kit ..
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The muscles used for locomotion in reside in the body wall. In the adult, there are 95 spindle shaped cells divided among four quadrants just underlying a basement membrane, hypodermis and cuticle. In each quadrant, the cells are arranged in interlocking pairs. In these muscle cells, myofilaments form a lattice that is restricted to a narrow zone of ~1.5 microns, just underlying the basement membrane and hypodermis. By polarized light microscopy, obvious striations are seen; bright (“birefringent”) A-bands alternate with dark I-bands; each I-band contains a row of dense bodies, which are the analogs of Z-discs of vertebrate striated muscle (). Because the striations lie at a slightly oblique angle with respect to the long axis of the worm, this muscle is called “obliquely striated”. Polarized light is also useful for evaluating the second largest set of muscles, those in the pharynx. Below we present a protocol for observing muscle using polarized light.

[35] Second-strand cDNA synthesis: mRNA fragments …

Following second strand synthesis, transfer contents of 0.5 ml microfuge into a 1.5 ml RNase-free microfuge tube.
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Because fixation is so critical for good ultrastructure, we present below several alternative protocols for TEM. At the end of the section we present a method for scanning EM (SEM) of worms. In this method, whole-mount animals or structures are viewed by EM, offering both broad-scale and highly resolved images ().

1977), fill-in of 5’ overhangs and removal of 3’ overhangs to form blunt ends (Sambrook 1989), and second strand synthesis in mutagenesis (Gubler 1987)Up:

MSDS NEBNext® Second Strand Synthesis Reaction Buffer.

Following second strand synthesis, transfer contents of 0.5 ml microfuge to 1.5 ml RNase-free microfuge tube.
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Shortly after entrance of the viral cores into the cytoplasm of a cell, the single-stranded plus-sense RNA genome of a retrovirus is converted into a double-stranded DNA molecule that subsequently integrates into the host cell genome (). This process, termed reverse transcription, requires two distinct RNA primers to synthesize the double-stranded DNA. The first primer is a host cell-derived tRNA that is used for the initiation of minus-strand DNA synthesis. The second is a short RNA derived by RNase H cleavages within a purine-rich sequence in the viral genome called the polypurine tract (PPT). This primer is used to begin plus-strand synthesis and is referred to as the plus-strand primer or the PPT primer (reference and references therein).

The NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module has been optimized to generate double stranded cDNA from first strand cDNA. The NEBNext Second Strand Synthesis Module is provided as a master mix to maximize efficiency and convenience in RNA sample preparation workflows.

The dsDNA generated by the NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module can be subsequently converted to blunt ended DNA fragments using the NEBNext End Repair Module.

The NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module has been validated by sequencing with the Illumina GAIIx in conjunction with M-MuLV (RNase H) Reverse Transcriptase, NEBNext First Strand Synthesis Reaction Buffer, Murine RNase Inhibitor, the NEBNext End Repair Enzyme Module, the NEBNext dA-Tailing Module, the NEBNext Quick Ligation Module, NEBNext High Fidelity 2x PCR Master Mix .

For larger volume requirements, customized and bulk packaging is available by purchasing through the OEM/Bulks department at NEB. Please contact for further information.

NEBNext ® Ultra II Non-Directional RNA Second Strand Synthesis ..
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E6106 NEBNext® First Strand Synthesis Reaction Buffer …

DTT stands for , a reducing agent. It probably is used to prevent the reverse transcriptase from oxidative damage.

From the looks of thing your cDNA synthesis method uses RNA replacement to synthesis the second strand. Basically after the reverse transcription (first strand synthesis), you have a RNA-DNA duplex. Treatment with the RNAse H during the second step, cuts up the RNA, leaving small fragment bound to the DNA strand. These bits of of RNA serves as primers for the synthesis of the second DNA strand.

FYI- ssDNA can loop back on itself froming a hair pin loop. The free end can thus act as a primer for the synthesis of the second DNA strand. However leaving things to chance is not the best of things.

First- and second-strand synthesis ..

Howdy all,

I'm generating cDNA from purified poly(A)RNA, following a protocol and noticed that there is no second strand primer. AND, the thermocycling conditions for the second strand are fairly cold - 2hrs @ 16C. Is it possible to have cDNA synthesis occur spontaneously without a primer? I'm wondering if this is a missing element in my protocol; the first strand primer (a polyT primer) shouldn't match the first strand synthesized, so what starts the second strand??? And what does the DTT do?

Thanks for any info!

Here's the scheme - most reagents are supplied by Invitrogen:

First Strand Synth -

polyA RNA
dT (polyT) primer

first strand buffer
DTT (what the heck is that?? comes with the superscript kit)
SuperScript III RT

Second Strand Synth
second strand buffer
DNA ligase
DNA polymerase
RNase H

T4 DNA polymerase

EDTA to stop rxn.

A putative primer for second-strand DNA synthesis of …

It is interesting to consider why initiation from the PPT primer at a nick with downstream RNA is intrinsically difficult. First, reverse transcriptase is much more facile at nondisplacement synthesis than displacement synthesis. Using DNA primers, nondisplacement synthesis is about 5- to 10-fold more efficient than displacement synthesis with a nontemplate DNA strand and at least 20-fold more efficient than displacement synthesis with a nontemplate RNA strand (, , , , , , ). In our experiments with RNase H-deficient reverse transcriptases, comparable differences were consistently observed between nondisplacement versus displacement synthesis in substrates containing the plus-strand RNA primer at a nick with downstream RNA. Second, reverse transcriptase extends DNA primers, including DNA versions of the PPT primer, much more efficiently than any RNA primers (, , , ). Third, the PPT assumes an unusual structure that might contribute to the difficulty in initiating plus-strand synthesis at a nick. This structural distortion promotes base unstacking and may render the majority of this sequence more resistant to RNase H degradation while facilitating RNase H cleavage at the −1/+1 site to generate the primer 3′ end (, , , ). The unusual structure of the PPT RNA-DNA hybrid does not require the binding of reverse transcriptase and is preserved whether the PPT sequence consists only of RNA or contains the RNA/DNA junction produced by plus-strand synthesis (). It is conceivable that the viral nucleocapsid protein facilitates the initiation of RNA displacement synthesis at a nick without any additional RNase H cleavages. Although our results do not exclude this possibility, in a previous study only a modest effect of nucleocapsid on RNA displacement synthesis was observed ().

Method for synthesis of the second strand of cDNA - …

Yet another possible benefit of a gap downstream of the PPT primer is that limited but sufficient extension from the plus-strand primer is permitted such that the nascent strand (instead of a less efficient RNA primer) is recognized as a preferred DNA 3′ terminus. The rate of polymerization for reverse transcriptase is affected by whether the primer strand is RNA or DNA (, ). Initiation of minus-strand synthesis has a distinct shift from a more distributive, slower mode of polymerization to a faster, more processive mode once the sixth nucleotide has been incorporated (, ). For plus-strand synthesis, significant pausing has been observed after a 1-nt extension and initiation becomes more efficient after the second nucleotide is incorporated (, ). Extension through a gap following the 3′ terminus of the PPT primer might allow reverse transcriptase sufficient distance to transition from the higher-fidelity mode of initiation to a more processive mode that extends the DNA 3′ terminus, which in turn promotes displacement of the downstream nontemplate strand. Consistent with this suggestion is the observation that DNA displacement synthesis with a DNA version of the PPT primer is much more efficient than that with an RNA PPT primer ().

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