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2x 1-Step RT-qPCR Mastermix (No ROX)

    Description

    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

    1. Thaw 1-Step 2X RT-PCR Master Mix and other reaction components at room temperature, mix each component, centrifuge and then place on ice.
    2. 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.
    3. Aliquot the assay mix into PCR tubes or plate.
    4. Add RNA template to PCR tubes or plate.
    5. Seal tubes with flat, optically transparent caps or seal plates with optically transparent film.
    6. Mix and then briefly centrifuge the tubes or plate.
    7. Program PCR instrument with indicated thermo-cycling protocol.
    8. Load PCR tubes or plate and start to run.
    9. 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, 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 

    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

    Product form

    SKU: RTQ01-00

    2x 1-Step RT-qPCR Mastermix (No ROX)

      Kit Features The kit is designed for singleplex RT-PCR with SYBR Green dye. For the reverse transcription step, this kit... Read more

      $180.00

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          Description

          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

          1. Thaw 1-Step 2X RT-PCR Master Mix and other reaction components at room temperature, mix each component, centrifuge and then place on ice.
          2. 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.
          3. Aliquot the assay mix into PCR tubes or plate.
          4. Add RNA template to PCR tubes or plate.
          5. Seal tubes with flat, optically transparent caps or seal plates with optically transparent film.
          6. Mix and then briefly centrifuge the tubes or plate.
          7. Program PCR instrument with indicated thermo-cycling protocol.
          8. Load PCR tubes or plate and start to run.
          9. 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, 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 

          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

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