myPOLS 6500 RT-PCR DNA Polymerase Instructions
- June 5, 2024
- myPOLS
Table of Contents
myPOLS 6500 RT-PCR DNA Polymerase Instructions
Contents
RevTaq RT-PCR DNA polymerase is supplied as a 50x solution containing glycerol together with an optimized 5x reaction buffer. An aptamer-based hot-start formulation prevents nonspecific amplification at ambient temperatures. Temperatures above 55°C cause the aptamer’s secondary structure to melt and will set free the polymerase.
Description
RevTaq RT-PCR DNA polymerase is an engineered, extremely thermostable reverse transcriptase and combined DNA polymerase, obtained through directed, artificial evolution. RevTaq RT-PCR DNA polymerase has a half-life at 95°C of
40 min.
RevTaq RT-PCR DNA polymerase allows “0-step” RT-PCRs directly from RNA templates (without an isothermal reverse transcription step), as reverse transcription takes place simultaneously with DNA amplification during the cycled PCR elongation step. This also facilitates reverse transcription reactions at high temperatures, thus minimizing the problems encountered with strong secondary structures in RNA that melt at elevated temperatures.
Quality Control
RevTaq RT-PCR DNA polymerase is tested for a successful, reproducible RT-qPCR
performance. Enzyme concentration is determined by protein-specific staining.
The activity of the enzyme is monitored and adjusted to a specific DNA
polymerase activity using an artificial DNA template and DNA primer.
Additionally, a 151 bp fragment (HPRT1 mRNA) is amplified from an in-vitro
transcribed RNA strand by a real-time RT-PCR protocol using hydrolysis probes
and the linearity of amplification over a specified serial dilution is
demonstrated.
No human RNA or nuclease contaminations are detected.
Material Safety Data (MSDS)
According to OSHA 29CFR1910.1200, Australia [NOHSC:1005, 1008 (1999)] and the
EU Directives 67/548/EC, 1999/45/EC and 1272/2008 (CLP Regulation) any
products which do not contain more than 1% of a component classified as
dangerous or hazardous nor more than 0.1% of a component classified as
carcinogenic, do not require a MSDS. However, we recommend the use of gloves,
lab coats and eye protection when working with these or any other chemical
reagents. myPOLS Biotec takes no liability for damage resulting from handling
or contact with this product. This product is not hazardous, not toxic, not
IATArestricted. Product is not from human, animal or plant origin.
The source of the product is recombinant protein expression in E. coli. The
product is for research use only and may be used for in-vitro experiments
only.
myPOLS Biotec GmbH, Byk-Gulden-Str. 2, 78467 Konstanz, Germany, T +49(0)7531 122 965 00
Recommendations for PCR/ Reaction Setup
RT-PCR/PCR Mix
| Component | Volume | Final concentration |
|---|---|---|
| RevTaq DNA Pol (50x) | 0.5 µl | 1x |
| 5x Volcano reaction buffer (5x) | 5 µl | 1x |
| dNTPs (10 mM) | 0.625 µl | 250 µM |
| Primer forward (10 µM) | 1.25 µl | 500 nM (50-1000 nM) |
| Primer reverse (10 µM) | 1.25 µl | 500 nM (50-1000 nM) |
| Probe (10 µM) | x µl | 50-1000 nM |
| Template/Sample extract* | y µl | >0.1 ng (0.1-2500 ng) |
| Nuclease-free water | up to 25µl total reaction vol. |
Keep all components on ice. Spin down and mix all solutions carefully before
use.
Recommended template concentration should be 0.004 ng/µl – 0.1 µg/µl (of total
RNA or genomic DNA).
Important notes
- Due to the aptamer-based hotstart-formulation, RevTaq RTPCR DNA polymerase will yield better results with annealing and extension temperatures above 57°C.
- Since the enzyme is thermostable, it is recommended to design very high melting primers and probes (>65°C).
- It is recommended to optimize the temperatures of the annealing/extension steps by applying a temperature gradient during establishments.
- Higher temperatures lead to higher specificity of the PCR. The RT-cycles are usually run at higher temperatures than the PCR-cycles since DNA-primer: RNA-template hybrids generally have higher melting points than DNA-primer: cDNA template duplexes.
- Depending on the employed assay, the reverse transcription steps may be omitted (0-step RT-PCR).
- RevTaq RT-PCR DNA polymerase is engineered and optimized for an amplicon size between 60- 300 bp.
Suggested RT-PCR protocol
RT-cycling (10 cycles)
Denaturation| 95°C| 3 sec
Reverse transcription| 65-80°C| 60 sec
(This temperature is usually higher than the temperature during PCR stage)
Directly followed by PCR-cycling (35-50 cycles)
Denaturation| 95°C| 10 sec
Annealing/Extension| 58-75°C| 50 sec
It is recommended to optimize the temperatures of the annealing/extension
steps by applying a temperature gradient during establishments.
References
RevTaq RT-PCR DNA polymerase is based on:
1. Structure and Function of an RNA-Reading Thermostable DNA Polymerase.
Angew. Chem. Int. Ed., 2013; 52: 11935–11939.
Blatter, N., Bergen, K., Nolte, O., Welte, W., Diederichs, K., Mayer, J.,
Wieland, M. and Marx, A.
Licences/Patents/Disclaimers
It is for the purchaser’s own internal research use and may not be resold, modified or used for production and commercial purposes of any kind without an agreement with myPOLS Biotec. For information on obtaining additional rights, please contact: info@mypols.de.
References
Read User Manual Online (PDF format)
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