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Reverse Transcription / cDNA Synthesis
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.
1. It depends on how much starting material you have and the primers/reverse transcriptase that you use. But basically smears are normal. Even when the rRNA is not visible.
2. I have never compared both, but I presume both would also be smears on the gel, as both would still produce varying lengths of cDNA.
3. The smear is fine. Just use the cDNA in a PCR to check, or run a spectro.
4. I would say it depends how many templates the primers stick to. The primer annealing step of 70C for 5 min prior to addition of reverse transcriptase decides how many templates have primers. Since there are no cyclic temperatures, the MMLV can only continue producing cDNAs from mRNAs (at 37C or 42C) that have primers annealed. So basically it's one RNA one cDNA.
First-strand cDNA Synthesis for Quantitative RT-PCR - …
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...
I posted my question elsewhere but hadn't answer, I'd like to post it here and, sorry it seems stupid question, because I'm really confused !
I read in somwhere a thing which perplexed me about cDNA. I'd like to know if cDNA is a double stranded or a single stranded ?
I know that cDNA is the copy of mRNA by reverse transcriptase so, theorically, it would be a single stranded , right or I'm mistake ?
Thanks for your feedback
iScript cDNA Synthesis Kit | | Bio-Rad
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)???
The Flex kit contains the same qScript enzyme in an format. The user can use their own gene specific primers or either the randomers or oligo(dT) included separately in the kit. This kit is ideal for end-point RT-PCR applications with relatively long (500bp+) amplicons, nested RT-PCR applications or other situations where it is desirable to control the priming of the 1st-strand synthesis step.
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First Strand cDNA Synthesis Kit | 69001 - Merck Millipore
- Fast cDNA synthesis protocol
- Optimized for various downstream applications, including next generation sequencing
- High content of full length transcripts
MMLV Reverse Transcriptase 1st-Strand 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.
SensiFAST™ cDNA Synthesis Kit - Bioline
the image will depend on the type of primer used in the synthesis: Random hexamers will copy any RNA including ribosomal RNA, while oligo dTs will only copy poly-A-tailed mRNA.
First-strand cDNA Synthesis for Quantitative RT-PCR
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.
cDNA Synthesis Kits | Biocompare Editorial Article
The includes a reverse transcriptase and a specialized set of reagents designed to yield cDNA that is optimal for gene cloning, cDNA library creation and quantitative PCR amplification.
iScript cDNA Synthesis Kit | 생명 과학 연구 | Bio-Rad
The kit uses Moloney Murine Leukemia Virus Reverse Transcriptase, RNase H Minus (M-MLV RT (H–)) which is an RNA-dependent DNA polymerase that is used in cDNA synthesis with long RNA templates. The lack of RNase H activity is important in this application in that RNase H activity will start to degrade template during long incubation times which are required for producing long cDNAs. RNase H minus RT enables preparation of long cDNAs and libraries containing a high percentage of full-length cDNA.
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