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First Strand cDNA Synthesis Protocols (E6300) | NEB
First introduced in Japan, PrimeScript is now available to scientists worldwide. Over 3,700 peer-reviewed articles have been published in which PrimeScript Reverse Transcriptase was referenced in a variety of scientific applications such as gene expression, gene discovery, transcriptome analysis, miRNA expression and regulation, molecular evolution, virology and microbiology. PrimeScript RTase is a modified, recombinant MMLV (Moloney Murine Leukemia Virus) reverse transcriptase and is verified to have low levels of RNase H. Because of the excellent extension capability of PrimeScript RTase, cDNA synthesis reactions can be performed at a lower temperature (42°C), decreasing the risk of RNA degradation that can occur during conventional reactions performed at higher temperatures.
Easy and Fast Sensitivity:
Random hexamers and oligo-dT prime reverse transcription in an unbiased mannerand capture more difficult-to-detect genes. The MMLV reverse transcriptase,optimized magnesium concentration, and other buffer components maximize cDNAproduct yield. The first strand kit is the most compatible for use with our RT²Real-Time™ SYBR Green PCR master mixes and subsequent gene expression analysiswith the RT² Profiler™ PCR Arrays as part of the complete PCR Array System.
First-strand cDNA Synthesis for Quantitative RT-PCR - …
I think most people don't show it because they don't bother to check it, and it's generally not of high scientific interest. I certainly never waste my cDNA by running it on a gel, it's just too precious. I use it in PCR directly after RT with the assumtion that RT worked, and run several internal controls to assess the quality of the cDNA. (beta actin, etc.) I never have any problems.
The intensity of the smear depends on how much you load. Try to calculate how much RNA and cDNA you would expect to have in your sample, and then use this to estimate how bright you expect your band to be. (Hint: RNA and ssDNA don't bind EtBr strongly anyway, so I would never expect a bright smear.)
Random hexamers also generate shorter cDNAs, especially if their concentration is high, so the shape of the smears might be different.
Can I ask why you are so preoccupied with verifying the products of your RT? If you are doing normal RT-PCR, don't bother running a gel; just proceed directly to PCR and do some internal PCR controls. If you are preparing a cDNA library, it would be preferable to prepare radiolabeled first-strand cDNA, run an alkaline gel alongside a labelled ladder, and then do autoradiography to determine the integrity of the cDNA alone (you won't see RNA in the radiograph).
Good question. However, I think the number of polymerase molecules in a reaction are limiting in this respect, and I think that there are a number of reasons strand displacement won't contribute to a significant increase in the number or cDNA molecules. (No time to explain, I have to go do some experiments!)
Hope this helps a bit,
For most cases, such for RT-PCR, 3' or 5' RACE, we just use single stranded cDNA (the first strand cDNA), while in case of preparing the cDNA for library construction, or RDA/SSH analysis which might need additional second strand synthesis step for generate double strand cDNA.
First Strand cDNA Synthesis (No …
The contains all of the reagents needed not only for theconversion of RNA into first strand cDNA, but also for the removal of genomicDNA from the RNA in the same simple two-step 20-minute reaction. The kit alsoprovides greater control over RNA quality while providing the same level ofsensitivity as other sources of reverse transcriptase or first strand cDNAsynthesis kits. This product is specifically designed for maximizing theperformance of the RT² Profiler™ PCR Array System.
In PCR one primer binds to one single stranded piece of DNA (that is why the first step is always to dentature at 94C(or so) to seperate any double stranded molecules into single strands. In the case of the first-strand cDNA from an RT reaction only one primer binds in the first round... This means that the first round of amplification in an RT-PCR is not logrithmic you simply synthesize the second strand of cDNA. In subsequent rounds (round 2 to round n) there is logrithmic amplification because both the forward and reverse primers bind to the denatured double stranded template.
In the first round, I think it is the forward primer that binds... I think this is because mRNA=forward strand in the genome and the first strand cDNA=complement of mRNA (or is the same as the reverse strand in the genome)
Someone check my logic here, I am confusing myself... is that right that the mRNA is the complement of the reverse strand of genomic DNA??
ie: mRNA sequence=forward strand=5'-3' sequence in gDNA (just with U not T)???
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TruScript First Strand cDNA Synthesis Kit ..
The PrimeScript First Strand cDNA Synthesis Kit contains all of the reagents necessary for synthesis of first-strand cDNA from total or poly(A)+ RNA using PrimeScript Reverse Transcriptase. Choose this cDNA synthesis kit when you want to synthesize full-length cDNA with provided primers (select from oligo dT or random 6-mer primers, both included in the kit.) This cDNA synthesis kit is powered by PrimeScript Reverse Transcriptase, which has exceptionally strong strand displacement activity and efficiently synthesizes cDNA. PrimeScript RTase is robust, versatile and well-suited for applications requiring full-length cDNA such as cDNA library construction. This PrimeScript-based cDNA synthesis kit also works well with other cDNA-dependent techniques including RT-PCR, cDNA probe preparation and real-time quantitative RT-PCR (qRT-PCR or RT-qPCR). The PrimeScript First Strand cDNA Synthesis Kit is ideal for reverse transcription of many different RNA templates, including GC-rich templates and RNAs with high levels of secondary structure.
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while in most cases, cDNA, abbreviate of complementary DNA, is definited as a single-strand DNA molecule with a nucleotide sequence that is complementary to an RNA molecule, and is synthesised in the lab from mRNA by reverse transcription...
Tailing reaction and first-strand cDNA synthesis)
you need to differenciate 2 things :
cDNA in general is the DNA molecule corresponding roughly to the mRNA seuqence. So It's a double stranded molecule included in plasmid for expression in most cases.
RT PCR : the mRNA molecule is reverse transcribed in cDNA single strand. After this point RNase eliminates the mRNA molecule and you get a single stranded cDNA molecule. As the next step is quite classical PCR, a single strand is ok for the exp (and the first step, which corresponds to sysnthesis of the corrseponding second strand of a DNA molecule restores the real cDNA molecule.
To be right, cDNA is a double stranded molecule, but for convenience, cDNA is also used for designing the reverse transcribed molecule of the RTPCR. It should be named as half cDNA or single strand cDNA.
cDNA is a shorting name.
SuperScript™ First-Strand Synthesis System for RT-PCR …
PrimeScript Reverse Transcriptase has exceptionally strong strand displacement activity and enables efficient preparation of cDNA up to 12 kb in length. It is robust, versatile and well-suited for applications requiring full-length cDNA such as preparation of cDNA libraries and other techniques involving first strand cDNA synthesis (RT-PCR, preparation of cDNA probes, real-time quantitative RT-PCR). PrimeScript RTase can be used for performing a reverse transcription reaction with any RNA template including GC-rich templates and RNAs with high levels of secondary structure. This enzyme is a modified, recombinant MMLV (Moloney Murine Leukemia Virus) reverse transcriptase and is verified to be RNase H Minus. Because of the excellent extension capability of PrimeScript Reverse Transcriptase, preparation of cDNA can be performed at a lower temperature (42°C), decreasing the risk of RNA degradation that can occur during conventional reactions performed at higher temperatures.
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