QuantumScript™ HD 逆转录酶
¥0.00 – ¥10,972.00
SKU | OPTIONS | 价格 | |
---|---|---|---|
SSIII-50 | 2,000U, 200 U/µl | ¥821.00 | |
SSIII-100 | 10,000U, 200 U/µl | ¥2,011.00 | |
SSIII-200 | 50,000U, 200 U/µl | ¥4,961.00 | |
SSIII-300 | 100,000U, 200 U/µl | ¥7,447.00 | |
SSIII-400 | 200,000U, 200 U/µl | ¥10,972.00 | |
SSIII-OEM | Any Size | Please inquire |
- Description
- Additional information
- Documents
Description
Description:
QuantumScript™ HD Reverse Transcriptase is an engineered version with increased sensitivity, improved specificity and maximum thermostability. QuantumScript™ HD Reverse Transcriptase has been engineered to have a longer half life at 50°C, which enables its ability to process longer RNAs with more complexed secondary structures. Enhanced thermostability of this enzyme is obtained through re-engineered RNA-based DNA Polymerase domain and the fusion of a novel RNA-interacting surface domain at the RNase H domain site. The enzyme is purified to homogeneity to ensure high thermostability, specificity, fidelity, yield, and more full length cDNA synthesis that the premium reverse transcriptase provides. The optimal fist-strand cDNA synthesis temperature for this enzyme is 50°C, and it has a broad working temperature range from 37° C to 55° C, with cDNA product size from 100 bp to 12 Kb.
Source
E. coli
Concentration
200 u/μl
Storage Buffer
20 mM Tris-HCl (pH 7.5), 1 mM DTT, 0.05% (v/v)Triton X-100, 0.1 mM EDTA, 0.1 M NaCl and 50% (v/v) glycerol.
Reaction Buffer (5x)
250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2, and 50 mM DTT
Unit Definition
One unit of the enzyme incorporates 1 nmole of dTTP into acid-precipitable material in 10 minutes at 37˚C using poly (A):oligo (dT)25 as template-primer.
Quality Control
This enzyme has passed the quality control assays: SDS-PAGE analysis for purity, functional absence of endonuclease activities, functional absence of exonuclease activities, functional absence of protease activity.
Storage and Handling: -20° C
Protocol
First-Strand cDNA Synthesis
Materials to Be Supplied by the User
- RNAse Inhibitor (Cat.# RNIN-100, RNIN-200, RNIN-300)
- dNTP, 10mM (Cat.# dNTP-10M, dNTP-25M)
- Nuclease-Free Water
The following procedure uses 10 pg to 5 µg of total RNA or 10 pg to 500 ng of mRNA.
- In a sterile RNase-free microcentrifuge tube, add primers (200-500ng of oligo(dT)12-18, 50-250ng of random primers or 2 pmol of specific primers). Heat the tube to 70° C for 5 minutes and incubate on ice for 1 min to denature any possible secondary structures within the template. Spin briefly to collect the solution at the bottom of the tube.
- Add the following components to the annealed primer/template in the order shown.
Note: Do not alter the ratio of primer to mRNA.
5 µl 5X Reaction Buffer; 5 µl of 10mM dNTP mixture (10mM each dATP, dGTP, dCTP and dTTP)
25 units RNAse inhibitor
0.5 µl QuantumScript Reverse Transcriptase (200 u/μl)
Add nuclease-free water to the final volume of 25µl - Mix gently. For random primers, incubate tube at 25°C for 5 min. Perform first-strand synthesis at 55°C for 30-60 min. Reaction temperature may be optimized between 50°C-60°C for difficult template with high secondary structure.
- Inactivate the enzyme by incubation at 70°C for 15 min.
- When perform PCR amplification after step 4, removal of RNA is highly recommended prior to the PCR amplification to ensure the yield of PCR product. Addition of 2 units of RNase H (Cat. # RNHE-100, RNHE-200, RNHE-300) and 20 min incubation at 37°C is recommended for the removal of RNA. Standard protocols for second-strand synthesis may be found in reference 2.
Note: The 5X Reaction Buffer is compatible with enzymes used in a number of downstream applications. Typically there is no need for phenol extractions or ethanol precipitations using this protocol before any PCR amplification.
References
- Roth, M.J., Tanese, N. and Goff, S.P. (1985) Purification and characterization of murine retroviral reverse transcriptase expressed in Escherichia coli. J. Biol. Chem. 260, 9326–35.
- Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) In: Molecular Cloning: A; Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 8.64.
Additional information
OPTIONS | 2,000U, 200 U/µl, 10,000U, 200 U/µl, 50,000U, 200 U/µl, 100,000U, 200 U/µl, 200,000U, 200 U/µl, Any Size |
---|
Manual & Protocols
COA ( Certificate of Analysis )