The AMV LongAmp® Taq RT-PCR Kit combines two powerful mixes, AMV Enzyme Mix and LongAmp® Taq 2X Master Mix for 2-step RT-PCR applications. The two mixes require minimal handling during reaction setup and yet offer consistent and robust RT-PCR reactions.
AMV Enzyme Mix can carry out first strand cDNA synthesis reactions with a broader optimal reaction temperature from 37°C to 50°C. LongAmp Taq 2X Master Mix offers consistent and robust amplification from more than 14 kb cDNA products. Primer d(T)23VN is included for anchored priming at the polyA-tail region of messenger RNAs. Random Primer Mix is an optimized mix of hexamer and d(T)23VN primers, offering good coverage of RNA templates.
General Information for Successful cDNA Synthesis:
Intact RNA of high purity is essential for sensitive RT-PCR detection. RNA should have a minimum A260/A280 ratio of 1.7 or higher.
Either total RNA or mRNA can be used in the reverse transcription reaction. Total RNA is generally sufficient for most RT-PCR analyses. However, if desired mRNA can be easily obtained using a PolyA Spin mRNA Isolation Kit (NEB #S1560) or Magnetic mRNA Isolation Kit (NEB #S1550).
The amount of RNA required for detection depends on the abundance of the transcript of interest. In general 1 ng to 1 μg total RNA or 0.1–100 ng mRNA are recommended.
First Strand cDNA Synthesis Reaction
Denaturation of RNA and primer at 70°C for 5 minutes can remove secondary structures that may impede long cDNA synthesis. However, this step can be omitted in some cases (unpublished results).
We recommend incubation at 42°C for one hour for maximum yield and length. However, many targets can be detected after a much shorter incubation time. For example, 5 minutes incubation is enough for a 2 kb cDNA synthesis.
Higher reaction temperature up to 60°C can be used for difficult targets of high secondary structures.
Choice of Primers for Reverse Transcription
Oligo d(T) priming is preferred for most applications because it ensures that all cDNA copies terminate at the 3´ end of the mRNA and produces the longest contiguous cDNA. An anchored oligo-d(T) primer [d(T)23VN] forces the primer to anneal to the start of the polyA tail, thereby preventing priming at internal sites in the polyA tail (1). However, two other priming choices are possible if desired.
The Random Primer Mix is an optimized mix of hexamer and d(T)23VN primers. It provides random priming sites covering the entire RNA templates including both mRNAs and non-polyadenylated RNAs (such as ribosomal RNAs). The Random Primer Mix yields shorter cDNAs on average and can be used for the detection of multiple short RT-PCR products. Random Primer Mix offers good performance in a wide range of RNA templates.
When a gene-specific primer is used in a cDNA synthesis reaction, the cDNA product can be used only for amplification of that transcript. This priming method gives good results when the amount of RNA is limiting (below 10 ng) and only one particular cDNA is desired.
Recommended primer concentration:
PRIMER -- Final conc.
OLIGO d(T)23VN -- 5 μM
RANDOM PRIMER MIX -- 6 μM
SPECIFIC PRIMER -- 0.1–1 μM
Specific primers for PCR should be designed with the aid of a primer design computer program to achieve best results, such as PrimerSelect™ (DNAStar Inc, Madison, MI) and Primer3. To minimize the complication introduced by contaminating genomic DNA, use primers that span an exon-exon boundary of the mRNA.
Most targets can be efficiently amplified using 1/10 (2 μl out of 20 μl cDNA synthesis reaction) or much less of the cDNA product (2).
A final concentration of 0.2 μM for each primer is recommended for PCR; however, it can vary between 0.05 μM to 1 μM.
The extension step using LongAmp Taq 2X Master Mix is recommended at 65–68°C with an extension rate of 50 seconds per kb.
For GC rich targets, we recommend to include DMSO up to 10%.
Use of thin-wall 0.2 ml PCR tubes and a manual hot-start may increase the PCR sensitivity and yield. A manual hot-start is done by assembling reactions into tubes placed on ice. The reaction tubes are transferred to a PCR machine with a block preheated at 95°C. Upon placement of the tubes, the cycler is immediately started.
HighlightsSemi-quantitative expression analysis of mRNAs
Cloning from RNA
High yields for a broad range of RT-PCR products
The following reagents are supplied with this product:
Store at (°C) Concentration Nuclease-free Water LongAmp® Taq 2X Master Mix 2 X AMV Enzyme Mix -20 10 X AMV Reaction Mix 2 X Oligo d(T)23 VN -20 50 μM Random Primer Mix -20 60 μM
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