Ready to use solutions optimized for Probe real time PCR (Taqman, Molecular Beacon etc) Include the HotStar DNA Polymerase, dNTPs, ROX and the stabilizing agent and an optimized PCR buffer, ensuring PCR specifity and sensitivity. The ROX in the mix is used for normalization of the flurescent signal in the qPCR. Compatible with real time thermal cyclers for probe real time PCR.

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Ready to use solutions optimized for Probe real time PCR (Taqman, Molecular Beacon etc) Include the HotStar DNA Polymerase, dNTPs, ROX and the stabilizing agent and an optimized PCR buffer, ensuring PCR specifity and sensitivity. The ROX in the mix is used for normalization of the flurescent signal in the qPCR. Compatible with real time thermal cyclers for probe real time PCR.

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Features The kit is designed for multiplex qPCR with TaqMan probes. The kit uses Super Taq-Probe DNA polymerase specially engineered for TaqMan probes, which increases 5’ to 3’ exonuclease efficiency and produces S-shaped curve. Up to four pairs of gene-specific primers can be applied in one reaction. The concentrations of the primers and probes are variable depending on specific assays and thermocycling protocols (Table 1).  The preferable PCR product size is ≤150bp. The kit has three formulations of No ROX, Low ROX or High ROX concentrations for your choice (see Table 2). Intended Use The 2x Multiplex qPCR Master Mix is used for real-time qualitative and quantitative multiplex qPCR with TaqMan probes for up to four targets.  The master mix is a premixed, 2X concentrated solution that has all the components except for gene-specific primers, probes and templates. 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 week.  Setup Reaction and Thermocycling  Thaw 2x Master mix and other reaction components at room temperature, mix each component, centrifuge and then place on ice.    Set up reactions (Table 1).  Seal tubes with flat, optically transparent caps or seal plate 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 150-400nM TaqMan probesb Variable Variable Each 150-250nM DNA Templatec Variable Variable ≤500ng human genomic DNA/20μL 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. Each probe’s Tm should be 8-10°C higher than the primer’s Tm, preferably between 70-75°C. DNA 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 95°C 2 min 1 II 95°C 10 sec 35-40 II 60°C, signal acquisition 60 sec 35-40 Table 3. Three-Step Thermocycling Protocol Stage Temperature Period Number of Cycles I 95°C 1 min 1 II 95°C 10 sec 35-40 II 60°C 30 sec 35-40 II 68-72°C, signal acquisition 30 sec 35-40 Notes: The three-step thermocycling protocol in Table 4 increases overall polymerase activity by 50%, a more effective protocol than Table 3. The primer concentration used in Tables 3 and 4 is typically 0.15-0.2uM. 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. Table 4. Compatible Instruments qPCR Instrument ROX required by instrument Passive dye setup Bio-Rad® iQ™5, CFX96, CFX384, Opticon Roche 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 

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Features The kit is designed for multiplex RT-qPCR with TaqMan probes. For the reverse transcription step, this kit uses a highly efficient Thermstable Reverse Transcriptase, 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 complementary DNA strands on RNA templates from gene-specific primers, ≥1 unit per 20μL of reaction. The RTase reversely-transcribes single digit copies of target RNA molecules consistently. The kit also contains Taq-Probe DNA polymerase, specially engineered for TaqMan probes and generating S-shaped curves. Up to four pairs of gene-specific primers can be applied in one reaction. The concentrations of the primers and probes are variable depending on assay designs and thermocycling protocols (Table 1).  The preferable PCR product size is ≤150bp. The kit has three formulations of No ROX, Low ROX or High ROX concentration for your choice (Table 4). Intended Use The 2x 1-Step Multiplex RT qPCR Master Mix is used for real-time qualitative and quantitative multiplex qPCR with TaqMan probes for up to four targets.  The master mix is a premixed, 2X concentrated solution that has all the components except for gene-specific primers, probes and RNA templates. The kit can also be applied to extraction-free RNA of cultured cells. 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 week.  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.      Set up reactions (Table 1).  Seal tubes with flat, optically transparent caps or seal plate 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 150-400nM TaqMan probesb Variable Variable Each 150-250nM DNA Templatec Variable Variable ≤500ng human genomic DNA/20μL 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.   Each probe’s Tm should be 8-10°C higher than the primer’s Tm, preferably between 70-75°C.   DNA 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 10 min 1 II 95°C 1 min 1 III 95°C 10 sec 35-40 III 60°C, signal acquisition 60sec 35-40 Table 3. Three-Step Thermocycling Protocol Stage Temperature Period Number of Cycles I 60°C 10 min 1 II 95°C 1 min 1 III 95°C 10 sec 35-40 III 60°C 30 sec 35-40 III 68-72°C, signal acquisition 30 sec 35-40 Notes: The three-step thermocycling protocol in Table 4 increases overall polymerase activity by 50%, a more effective protocol than Table 3. The primer concentration used in Tables 3 and 4 is typically 0.15-0.2uM. 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. Table 4. Compatible Instruments qPCR Instrument ROX required by instrument Passive dye setup Bio-Rad® iQ™5, CFX96, CFX384, Opticon, Roche 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 

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