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In vitro destabilization of plant viruses and cDNA synthesis

transcriptase with reduced RNase H activity because this results in greater full-length cDNA synthesis.

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Synthesis of a cDNA Library Using Small Amounts of …

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
dNTPs
water


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



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

T4 DNA polymerase

EDTA to stop rxn.

and subsequent cDNA synthesis utilizing the 'built-in' primer contained ..

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.

RNA-Quant cDNA synthesis overview The initial polyadenylation ..

The objective of this research is to investigate the effect of the reducing agent dithiothreitol () on enhancing ethanol production from synthesis gas (syngas) using Clostridium strain P11 in 250-mL serum bottles. Reducing agents help in regeneration of NADH from NAD+. NADH is utilized in the production of alcohol from aldehydes. The effect of DTT concentrations from 0 to 10 g/L was studied in 1.0 g/L yeast extract (YE) and 10 g/L corn steep liquor (CSL) media and with simulated syngas and producer gas. Syngas contains mainly carbon monoxide, hydrogen, carbon dioxide and nitrogen. The fermentation process was followed for 360 h. Liquid samples were collected every 24 h to determine cell mass, pH and product concentrations. The experiment was done in quadruplets at each DTT concentration and the results were analyzed for statistical significance using SAS version 9.2 at 95% confidence level. Results showed that over 350% increase in ethanol concentration was obtained in media that contained at least 7.5 g/L of DTT in the 1.0 g/L yeast extract medium after 360 h of fermentation with simulated syngas compared to the control medium (without DTT). However, only a 35% increase in ethanol production was noticed in 10 g/L corn steep liquor media in the presence of 2.5 and 5.0 g/L of DTT compared to the control medium with simulated syngas. In addition, DTT (at a concentration of 2.5 g/L) produced about 240% more butanol in the 10 g/L CSL medium compared to the control with simulated syngas. The results suggested that the use of small concentrations of DTT in the broth enhances ethanol production from simulated syngas in YE media. When producer gas was used, DTT enhanced isopropanol production instead of ethanol production in both YE and CSL media. The electrons donated by might have been utilized in the reduction of acetone to isopropanol by strain P11 instead of reduction of acetaldehyde to ethanol. The removal of acetone and other impurities from the producer gas could enhance DTT effectiveness as a reducing agent and improve ethanol production in both YE and CSL media. The objective of this research is to investigate the effect of the reducing agent dithiothreitol (DTT) on enhancing ethanol production from synthesis gas (syngas) using Clostridium strain P11 in 250-mL serum bottles. Reducing agents help in regeneration of NADH from NAD+. NADH is utilized in the production of alcohol from aldehydes. The effect of DTT concentrations from 0 to 10 g/L was studied in 1.0 g/L yeast extract (YE) and 10 g/L corn steep liquor (CSL) media and with simulated syngas and producer gas. Syngas contains

M-MuLV Reverse Transcriptase synthesizes a complementary DNA strand initiating from a primer using either RNA (cDNA synthesis) or single-stranded DNA as a template.

What is the function of magnesium chloride in complementary DNA ..

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    From the looks of thing your cDNA synthesis method uses RNA replacement to synthesis the second strand.

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    In the one-step approach, the entire reaction from cDNA synthesis to PCR amplification occurs in a single tube.

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    dT for priming cDNA synthesis

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