One-Step RT-PCR Kit provides reverse transcription and subsequent PCR in the same reaction tube, eliminating the need to change buffers and reducing the risk of contamination. This kit offers the end-user an easy alternative to the conventional “two-step” RT-PCR approach with extreme sensitivity, specificity and high product yield.  Composition:  SuperHiFi™ DNA polymerase (5U/µl), 25µl PowerScript™ Reverse Trascriptase (200U/µl), 25µl 2xOne-Step RT-PCR Buffer, 1ml Applications Gene-expression analysis Transcription analysis Gene cloning Multiplex RT-PCR High throughput applications Virus detection and quantification

Read more less

One-Step RT-PCR Kit provides reverse transcription and subsequent PCR in the same reaction tube, eliminating the need to change buffers and reducing the risk of contamination. This kit offers the end-user an easy alternative to the conventional “two-step” RT-PCR approach with extreme sensitivity, specificity and high product yield.  Composition:  SuperHiFi™ DNA polymerase (5U/µl), 25µl PowerScript™ Reverse Trascriptase (200U/µl), 25µl 2xOne-Step RT-PCR Buffer, 1ml Applications Gene-expression analysis Transcription analysis Gene cloning Multiplex RT-PCR High throughput applications Virus detection and quantification

Read more less

A novel recombinant reverse transcriptase that exhibits much higher efficiency in the first-strand cDNA synthesis from RNA templates with secondary structures and high GC content. PowerScriptTM Plus Reverse Transcriptase is engineered to perform under high temperatures (50°C - 60°C), facilitating the elimination of secondary structures associated with GC-rich RNA templates. Due to this unique feature, ThermoScriptTM Plus can synthesize full-length cDNA libraries from RNA templates up to 12 kb in length.  Features  Lack RNase H activity: higher yield of cDNA.  Thermal stable: the optimum reaction temperature is 50°C, the highest is 60°C. Can overcome the template RNA secondary structure, and finish the reverse transcriptase experiment smoothly.  Wide temperature range: can reverse transcript from 37-60°C, with more than 80% of the highest activity at 42°C-60°C. customer can choose the reaction temperature freely.  Strong amplification activity: Gene mutation enhanced the binding capacity of the enzyme and RNA. Increased the amplification speed, can obtain the quality cDNA, suitable for cDNA library construction.  Applications Synthesizing cDNA from a ssRNA Constructing cDNA library Source Recombination of E. coli containing Moloney murine leukemia virus reverse transcriptase gene from clone of Moloney murine. Unit definition Using Poly (A) as the template and oligo (dT) as the primer, the enzyme required to catalyze the incorporation of 1 nmol of dTTP within 10 minutes at 37 oC is defined as one active unit (U). Concentration: 200 U/ul Size: 25 ul Form: Enzyme supplied with 5X RT buffer  Storage: Store all components at -20°C

Read more less

Kit Features The kit is designed for singleplex RT-PCR with SYBR Green dye. For the reverse transcription step, this kit uses a highly efficient Thermophilic Reverse Transcriptase, which is a thermophilic type A polymerase, with optimal temperatures of 60-62°C. The RTase is easily heat-inactivated at ≥90°C for 1min. The RTase efficiently synthesizes a complementary DNA strand on RNA template from a gene-specific primer, ≤1 unit per 20μL of reaction. The RTase reversely-transcribes single digit copies of target RNA molecules consistently. The kit also contains Taq DNA polymerase for PCR with SYBR Green dye. The concentrations of the primers are variable depending on assay designs and thermocycling protocols (Table 1). The preferable PCR product size is ≤150bp. The kit has three formulations of ROX, Low ROX or High ROX concentrations for your choice.  Transportation and Storage The kit can be transported at below 4°C for up to 3 days. The kit should be kept stable in the dark at -20°C for ≤24 months with ≤10 times of freeze-thaw cycles. The kit can be stored at 4°C for a weak. Setup Reaction and Thermocycling Thaw 1-Step 2X RT-PCR Master Mix and other reaction components at room temperature, mix each component, centrifuge and then place on ice. Determine the total volume for the number of reactions, add 5-10% extra volume, and prepare assay mix of all components except RNA template. Mix the assay mix, centrifuge and then place on ice. Aliquot the assay mix into PCR tubes or plate. Add RNA template to PCR tubes or plate. Seal tubes with flat, optically transparent caps or seal plates with optically transparent film. Mix and then briefly centrifuge the tubes or plate. Program PCR instrument with indicated thermo-cycling protocol. Load PCR tubes or plate and start to run. Perform data analysis according to the PCR instrument instructions. Table 1. Setting up a 20μl or 10μl Reaction Component Volume per 20μl Volume per 10μl Final Concentration 2x Mastermix 10μL 5μL 1X Primersa Variable Variable Each 150-900nM RNA Templatesb Variable Variable As low as single digit copies of target RNA to ≤1μg total RNA H2O To 20μL To 10μL Note: Each primer’s Tm should be designed ≥60°C, preferably between 62°C to 65°C, using primer3 software for high efficiency and specificity.   RNA templates should be extracted by a qualified silica-based kit and eluted with low EDTA TE buffer (10mM Tris-HCl, 0.1mM EDTA, pH 8.0-8.3). Table 2. Standard Thermocycling Protocol Stage Temperature Period Number of Cycles I 60°C 10min 1 II 95°C 1min 1 III 95°C 10sec 35-40 III 60°C, signal acquisition 60sec 35-40 IV 60°C to 95°C Various 1 Table 3. Three-Step Thermocycling Protocol Stage Temperature Period Number of Cycles I 60°C 10min 1 II 95°C 1min 1 III 95°C 10sec 35-40 III 60°C 30sec 35-40 III 68-72°C, signal acquisition 30sec 35-40 IV 68°C to 95°C Various 1 Notes: The three-step thermocycling protocol in Table 3 increases overall polymerase activity by 50%, a more effective protocol than Table 2. The primer concentration used in Tables 2 and 3 is typically 0.15-0.2uM. Table 4. Compatible Instruments qPCR Instrument ROX required by instrument Passive dye setup Bio-Rad® iQ™5, CFX96, CFX384, OpticonRoche Lightcycler®Qiagen Rotor-Gene™Eppendorf Mastercycler®Cepheid® SmartCycler® Not recommended Not necessary Applied Biosystems® 7500, 7500 Fast, QuantStudio™, ViiA7™, Agilent Mx™ Low ROX(50nM final concentration) Turn on ROX passive reference dye  Applied Biosystems® 5700, 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne™, StepOnePlus™ High ROX(500nM final concentration) Turn on ROX passive reference dye  Precautions If you order a “No ROX” master mix but you have an Applied Biosystems or Thermo Fisher instrument, please turn off ROX passive reference dye button when setup assays.

Read more less

A novel recombinant reverse transcriptase that exhibits much higher efficiency in the first-strand cDNA synthesis from RNA templates with secondary structures and high GC content. PowerScriptTM Plus Reverse Transcriptase is engineered to perform under high temperatures (50°C - 60°C), facilitating the elimination of secondary structures associated with GC-rich RNA templates. Due to this unique feature, ThermoScriptTM Plus can synthesize full-length cDNA libraries from RNA templates up to 12 kb in length.  Features  Lack RNase H activity: higher yield of cDNA.  Thermal stable: the optimum reaction temperature is 50°C, the highest is 60°C. Can overcome the template RNA secondary structure, and finish the reverse transcriptase experiment smoothly.  Wide temperature range: can reverse transcript from 37-60°C, with more than 80% of the highest activity at 42°C-60°C. customer can choose the reaction temperature freely.  Strong amplification activity: Gene mutation enhanced the binding capacity of the enzyme and RNA. Increased the amplification speed, can obtain the quality cDNA, suitable for cDNA library construction.  Applications Synthesizing cDNA from a ssRNA Constructing cDNA library Source Recombination of E. coli containing Moloney murine leukemia virus reverse transcriptase gene from clone of Moloney murine. Unit definition Using Poly (A) as the template and oligo (dT) as the primer, the enzyme required to catalyze the incorporation of 1 nmol of dTTP within 10 minutes at 37 oC is defined as one active unit (U). Concentration: 200 U/ul Size: 25 ul Form: Enzyme supplied with 5X RT buffer  Storage: Store all components at -20°C

Read more less

Two Step RT-PCR Kit consists of two main components: PowerScript™ Plus cDNA Synthesis MasterMix for reverse transcription 2XTaqPlus PCR MasterMix for subsequent PCR Composition: PowerScript™ Plus Reverse Transcriptase (200U/µl, 30µl) 2xReaction mix contains Ribonuclease Inhibitor, dNTPs, Oligo(dT)s and Random Primers, 350µl Nuclease-free H2O, 1ml  2XTaqPlus PCR MasterMix, 1ml Applications Gene-expression analysis Transcription analysis Gene cloning Multiplex RT-PCR High throughput applications Virus detection and quantification

Read more less