2x 1-Step Multiplex RT-qPCR Mastermix for TaqMan Probe (No ROX)

Description

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 

  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. Set up reactions (Table 1). 
  3. Seal tubes with flat, optically transparent caps or seal plate with optically transparent film.
  4. Mix and then briefly centrifuge the tubes or plate.
  5. Program PCR instrument with indicated thermo-cycling protocol.
  6. Load PCR tubes or plate and start to run.
  7. 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 

Read more

Product form

SKU: RTQM01-00

2x 1-Step Multiplex RT-qPCR Mastermix for TaqMan Probe (No ROX)

Features The kit is designed for multiplex RT-qPCR with TaqMan probes. For the reverse transcription step, this kit uses a... Read more

$180.00 Excl. VAT

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    Description

    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 

    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. Set up reactions (Table 1). 
    3. Seal tubes with flat, optically transparent caps or seal plate with optically transparent film.
    4. Mix and then briefly centrifuge the tubes or plate.
    5. Program PCR instrument with indicated thermo-cycling protocol.
    6. Load PCR tubes or plate and start to run.
    7. 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 

    Read more

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