Description
General description
The First Strand cDNA Synthesis Kit is used for the synthesis of the first strand cDNA as the starting reaction for two-step RT PCR.The kit includes Reverse Transcriptase AMV for first strand synthesis, two different primers, our PCR Nucleotide Mix, and Control Neo pa RNA. PCR products that are generated by RT-PCR can be cloned using standard procedures.
The amplification of RNA requires the conversion of the RNA substrate into DNA. This is achieved through the use of a reverse transcriptase such as AMV RT (avian myeloblastis virus reverse transcriptase) or M-MuLV RT (moloney murine leukemia virus reverse transcriptase). The resulting cDNA can be used as a template for a standard PCR.
AMV RT synthesizes the new cDNA strand at site(s) determined by the type of the primer used:
at the 3′-end of the poly(A) mRNA when Oligo-p(dT)₁₅is used as a primer,
at nonspecific points along the mRNA template when using the random primer p(dN)₆, or
at a site determined by a sequence-specific primer.
The use of AMV RT for first-strand cDNA synthesis has certain advantages. The higher thermostability of this enzyme compared to M-MuLV RT allows the reaction to be performed at +42°C, giving higher specificity and better resolution of secondary structures compared to a reaction performed at +37°C.
Application
The First Strand cDNA Synthesis Kit for RT-PCR (AMV) is suitable for:
Detection of the presence or absence of RNA viruses or other RNA-containing microorganisms (in combination with PCR)
Quantification of mRNA for monitoring differential expression of a specific mRNA
First step in the "differential display of mRNA"
Generation of cDNA libraries with large and full-length inserts
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for reverse transcription of RNA into complementary DNA.
Biochem/physiol Actions
Heat inactivation: 5 min, 95 °C
Reverse Transcriptase AMV
Features and Benefits
Robustness:
Transcribes total RNA, mRNA and viral RNA along with difficult-to-transcribe secondary RNA structures
Obtain cDNA transcripts up to 12 kb
Higher thermostability (up to 50°C) and specificity than M-MuLV Reverse Transcriptase
Purification of the resulting cDNA before PCR reaction is not necessary
Flexibility:
It can be used with either sequence-specific primers, poly(dT)15 primers, or random primers, p(dN)6
Packaging
1 kit containing 10 components.
Analysis Note
In the standard cDNA synthesis assay, at least 300 ng of cDNA is synthesized (i.e., 30% yield) when 1.0 μg Neo pa RNA template is incubated with 20 μCi [α-³²P]-dCTP (specific activity of 3000 Ci/mmol) for 60 minutes at +42°C.
Other Notes
For life science research only. Not for use in diagnostic procedures.
Volume Activity: 20 to 25 U/μl
