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Finally, a double stranded cDNA is synthesized.
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,
The takes advantage of a linker‐primer consisting of (in order from 3' to 5') an oligo(dT) primer, a restriction site for the XhoI endonuclease, and a (GA)20 repeat to protect the restriction site during generation of the blunt‐ended cDNA.
First cycle, first strand cDNA synthesis
The reverse transcriptase uses the free end and synthesizes a single stranded cDNA in the presence of dCTP, dGTP, dATP and dTTP, and results in mRNA-cDNA hybrid.
Set up reverse transcription rxns using 200ng of each DNased RNA, using Promega oligo dT primers and M-MLV Reverse Transcriptase according to Promega protocol. . Primer and DNAsed RNA were mixed and brought up to 18.25uL with H2O. Samples were heated @ 70C for 5mins and then placed immediately on ice. . 6.75uL of the RT master mix was added to each tube, mixed, spot spun and then incubated @ 42C for 1hr, heat inactivated @ 95C for 3mins and stored @ 4C.
Second Cycle, first strand cDNA synthesis
Both poly(A) + mRNA and total RNA can be used for first-strand cDNA synthesis, but poly(A) + mRNA
may give higher yields and improved purity of final products.
The newly synthesized firststrand cDNA is ready for immediate downstream applications, or for long-term storage
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Oligo (dT) 18 Primer Random Hexamer Primers
**We use MMLV reverse transcriptase but there are other RTs that vary in their efficiency, etc. The important thing, if you are planning to use these cDNAs for quantification and comparison across tissues or individuals, is to be consistent and always use the same RT and cDNA synthesis conditions.
Oligo-dT Primers - QIAGEN Online Shop
- Fast cDNA synthesis protocol
- Optimized for various downstream applications, including next generation sequencing
- High content of full length transcripts
cDNA Library Construction Kits - SMART cDNA Synthesis
Mint-2 cDNA synthesis kit is designed to synthesize full-length-enriched double stranded (ds) cDNA from total or poly(A)+ RNA. Synthesized cDNA can be used for construction of cDNA libraries, subtractive hybridization (SSH), high-throughput sequencing on the next generation sequencing platforms (Roche/454, ABI/SOLiD or Illumina/Solexa), and other applications.
sequence and modified oligo dT primer are not added onto genomic ..
cDNA synthesis method utilizes a specific feature of the Moloney murine leukemia virus reverse transcriptase (RT). First-strand cDNA synthesis starts from the 3'-end CDS adapter containing an oligo(dT) sequence that anneals to poly(A) stretches of RNA. When RT reaches the 5'-end of the mRNA, it adds several non-template nucleotides, primarily deoxycytidines, to the 3'-end of the newly synthesized first-strand cDNA (Schmidt and Mueller, 1999; Matz , 1999). This oligo(dC) stretch base anneals to a complementary oligo(dG) sequence located at the 3'-end of a special deoxyribooligonucleotide adapter, called PlugOligo. Mint RT identifies PlugOligo as an extra part of the RNA template and continues first strand cDNA synthesis to the end of the oligonucleotide, thus incorporating PlugOligo sequence into the 5'-end of cDNA.
a proprietary adapter oligo(dT) primer
*You could also use plain oligo dT, but using the GR version adds an adaptor that allows you to do 3'RACE with the GR-3' primer. And it can't hurt to use it even if you won't do 3'RACE.
Protocol cDNA synthesis in oligo (dT)25 magnetic …
The synthesis of DNA from an RNA template, via reverse transcription, produces complementary DNA (cDNA). Reverse transcriptases (RTs) use an RNA template and a short primer complementary to the 3' end of the RNA to direct the synthesis of the first strand cDNA, which can be used directly as a template for the Polymerase Chain Reaction (PCR). This combination of reverse transcription and PCR (RT-PCR) allows the detection of low abundance RNAs in a sample, and production of the corresponding cDNA, thereby facilitating the cloning of low copy genes. Alternatively, the first-strand cDNA can be made double-stranded using DNA Polymerase I and DNA Ligase. These reaction products can be used for direct cloning without amplification. In this case, RNase H activity, from either the RT or supplied exogenously, is required.
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