2x Fast qPCR Master Mix (High ROX)

Description

Intended Use

  • The 2x Fast qPCR Mastermix is used for real-time qualitative and quantitative qPCR with SYBR Green dye. ;
  • The Mastermix is a premixed, 2x concentrated solution that has all the components except for gene-specific primers, probes and templates

Kit Characterizations

  • The kit is designed for a single gene qPCR with SYBR Green dye.
  • The kit contains Taq-Fast DNA polymerase which can extend more than 300 bases with a short cycling program.
  • 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 corresponding instruments(see Table 4).

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 2x Mastermix 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 150-900nM
DNA Templateb Variable Variable ≤30ng genomic DNA/10μL
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.
  • 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 12 sec 35-40
II 60°C, signal acquisition 60 sec 35-40
III 60°C to 95°C  Various 1

Note: The primer concentration used is typically 0.2uM

Table 3. Fast Thermocycling Protocol 

Stage Temperature
 Period Number of Cycles
I 95°C 1 min 1
II 95°C 5 sec 35-40
II 60°C, signal acquisition 30 sec 35-40
III 60°C to 95°C  Various 1

Note: 

  • The product size for the fast thermocycling protocol is preferred to be less than 90bp.
  • The primer concentration used is typically between 0.4uM and 0.9uM.

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 

 

Quality Control 

Not detectable DNase and RNase contaminations. 

Precautions 

If you order a “No ROX” master mix but you have an Applied Biosystems or ThermoFisher instrument, please turn off ROX passive reference dye button when setup assays.

Read more

Product form

SKU: QP13-00

2x Fast qPCR Master Mix (High ROX)

Intended Use The 2x Fast qPCR Mastermix is used for real-time qualitative and quantitative qPCR with SYBR Green dye. ;... Read more

$70.00 Excl. VAT

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    Description

    Intended Use

    • The 2x Fast qPCR Mastermix is used for real-time qualitative and quantitative qPCR with SYBR Green dye. ;
    • The Mastermix is a premixed, 2x concentrated solution that has all the components except for gene-specific primers, probes and templates

    Kit Characterizations

    • The kit is designed for a single gene qPCR with SYBR Green dye.
    • The kit contains Taq-Fast DNA polymerase which can extend more than 300 bases with a short cycling program.
    • 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 corresponding instruments(see Table 4).

    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 2x Mastermix 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 150-900nM
    DNA Templateb Variable Variable ≤30ng genomic DNA/10μL
    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.
    • 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 12 sec 35-40
    II 60°C, signal acquisition 60 sec 35-40
    III 60°C to 95°C  Various 1

    Note: The primer concentration used is typically 0.2uM

    Table 3. Fast Thermocycling Protocol 

    Stage Temperature
    		 Period Number of Cycles
    I 95°C 1 min 1
    II 95°C 5 sec 35-40
    II 60°C, signal acquisition 30 sec 35-40
    III 60°C to 95°C  Various 1

    Note: 

    • The product size for the fast thermocycling protocol is preferred to be less than 90bp.
    • The primer concentration used is typically between 0.4uM and 0.9uM.

    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 

     

    Quality Control 

    Not detectable DNase and RNase contaminations. 

    Precautions 

    If you order a “No ROX” master mix but you have an Applied Biosystems or ThermoFisher instrument, please turn off ROX passive reference dye button when setup assays.

    Read more

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