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Description
Formulated for highly reproducible first-strand cDNA synthesis and subsequent PCR in a single tube.
Product Highlights
Sensitive – incorporates a blend of high-affinity RT and novel MyTaq HS DNA Polymerase, enabling amplification of low-copy number targets from ≥3 pg total RNA
Efficient – novel one-step buffer system maximizes the efficiency of both the reverse transcription and PCR steps, delivering improved yield of any target
Robust – RT tolerates the higher reaction temperatures required to overcome secondary structure, giving reliable detection of even challenging and GC-rich targets
Specific – MyTaq HS DNA Polymerase is an antibody-mediated hot-start enzyme that remains completely inactive during PCR set-up to prevent non-specific amplification
Flexible – utilizes gene-specific primers for full-length reverse transcription and subsequent PCR amplification of any RNA target
Convenient – an all-in-one-tube mastermix that improves the speed, convenience and accuracy of RT-PCR
Product Description
MyTaq™ One-Step RT-PCR Kit incorporates the latest advances in buffer chemistry, including Meridian ultra-pure dNTPs, together with a proprietary reverse transcriptase and MyTaq HS, a new generation of antibody-mediated hot-start DNA polymerase. This ensures that MyTaq One-Step RT-PCR Kit enables fast, highly-specific and ultra-sensitive amplification of RNA targets for use in a broad range of downstream applications.
MyTaq One-Step Kit is ideal for determining the presence or absence of RNA templates and quantifying expression through qualitative or semi-quantitative analysis of RNA transcription levels. The one-step format uses gene specific primers to maximize amplification of these targets, whilst eliminating non-specific amplification, ensuring highly efficient and sensitive transcription from as little as 3 pg total RNA.
MyTaq One-Step RT-PCR Kit is perfect for the synthesis of double-stranded cDNA products for subsequent gene expression analysis over a broad temperature range, the use of higher temperatures allows reverse transcription through RNA secondary structure, including difficult and GC-rich sequences. Enhanced amplification of these targets ensures high cDNA yields from all RNA, including total RNA, mRNA, in vitro transcribed RNA, snRNA and viral RNA.
Applications
Gene-expression analysis
Transcription analysis
cDNA cloning
Multiplex RT-PCR
A 1 kb fragment was amplified in duplicate from serial dilution of mouse total RNA (30 ng, 3 ng, 300 pg, 30pg and 3 pg respectively) using RN18S-1000 primers and the MyTaq One-Step RT-PCR Kit. The completed RT-PCR reactions were analysed on a 1% agarose gel. HyperLadder 50bp (M). The results show that the MyTaq One-Step RT-PCR Kit achieves high–yield, specific amplification from even complex templates.
A 1 kb fragment was amplified in duplicate from a serial dilution of mouse total RNA (10 ng, 2 ng, 400 pg, 80 pg, 16 pg and 3 pg; lanes 1-6 respectively) using RN18S-1000 primers and the MyTaq One-Step RT-PCR Kit. HyperLadder 50bp (M). The reverse transcriptase in the MyTaq One-Step RT-PCR Kit was able to deliver high quality cDNA even at 50°C, over a broad dynamic range.
Introduction to MyTaq
Overview, features and benefits of the MyTaq product family
When we compared the performance of our routine supplier’s RTase against Meridian’s MyTaq One-Step RT-PCR, the other supplier’s RTase gave two false negatives in five different grapevine samples tested for Grapevine rupestris stem-pitting-associated Foveavirus. We were convinced to immediately switch.
All components should be stored at -20°C upon receipt for optimum stability. Repeated freeze/thaw cycles should be avoided.
When stored under the recommended conditions and handled correctly, full activity of the reagents is retained until the expiry date indicated on the outer box label.
""When we compared the performance of our routine supplier’s RTase ...
"
University of Adelaide, Australia
"When we compared the performance of our routine supplier’s RTase against Meridian’s MyTaq One-Step RT-PCR, the other supplier’s RTase gave two false negatives in five different grapevine samples tested for Grapevine rupestris stem-pitting-associated Foveavirus. We were convinced to immediately switch."
In a one-step reaction the reverse transcription reaction and the real-time PCR reaction are done in one tube, making this a closed tube assay, so contamination can be avoided. It saves pipetting steps and time and is easy in handling, making it ideal for high throughput screening.
In a two-step reaction the reverse transcription reaction and the real-time PCR reaction are done in separate tubes. It gives a more flexible way of working in that the cDNA can be used for more than one real-time PCR reaction and can be archived, eliminating the need to continually isolate RNA. For convenience Meridian sells the SensiFAST cDNA Synthesis Kit separately if you wish to take a two-step approach.
PCR can be a challenging technique, with various parameters to optimize to achieve the best results. If you are having problems, these could be easily resolved by addressing a few issues. Please see our PCR troubleshooting guide for suggestions and help with your specific problems.
Observation
Recommended Solution(s)
No or low PCR yield
Enzyme concentration too low – increase the amount of enzyme in 0.5 U increments.
Primers degraded – check quality and age of the primers.
Magnesium concentration too low – increase concentration in 0.25 mM increments with a starting concentration of 1.75 mM.
Primer concentration not optimized. Titrate primer concentration (0.3-1 µM); ensuring that both primers have the same concentration.
Template concentration too low – Increase concentration of template.
Perform a positive control to ensure that the enzyme, dNTPs and buffers are not degraded and/or contaminated.
Multiple Bands
Primer annealing temperature too low. Increase annealing temperature. Primer annealing should be at least 5°C below the calculated Tm of primers.
Prepare master mixes on ice or use a heat-activated polymerase.
For problems with low specificity. Try adding 3% DMSO (not supplied) to improve specificity.
Smearing or artifacts
Template concentration too high. Prepare serial dilutions of template.
Too many cycles. Reduce the cycle number by 3-5 to remove non-specific bands.
Enzyme concentration too high - decrease the amount of enzyme in 0.5 U increments.
Extension time too long. Reduce extension time in 0.5-1 minute increments.
During PCR setup at room temperature, standard polymerases will have some activity. When using a hot-start polymerase, this enzyme will have no activity at room temperature, thus reducing the risk of unspecific products in the reaction due to these mis-primed oligonucleotides. This feature makes this type of polymerase ideally suited to high-throughput applications, where the reactions may be left sitting at room temperature for prolonged periods of time.
