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EnoGene Green qPCR Master Mix
Datasheet   

  • Catalog Number:

    E31M001

  • Amount:

    500reactions

  • Price:

    75$

  • Storage/Stability:

    This reagent can be stored at 2-8°C for 6 months and protected from light. For longer storage, this reagent should be kept at -20°C and protected from light. 

  • Experimental Methods:

    COMPONENTS:
    E31M001 can be used for 500 reactions for a total 20µL reaction volume.
    Cat NO. Components Size
    E31M001 2xqPCR Master Mix   1 mL ×5tubes

    PRIMER DESIGN
    - Primer length: 20~30bp
    - GC content of primer: 40~80%
    -Target length: ≤ 200 bp (optimally, ≤ 150bp) 

    SPECIMEN:
    -cDNA :  Reverse transcription reactions from total or poly (A)+ RNA may be used directly, or after dilution, for realtime PCR. Purified RNA by phenol/ chloroform extraction and ethanol precipitation may also be used.  Oligo dT and random primers  are suitable for the reverse transcription reaction.
    -Genomic DNA:  Purified DNA, which would be used for general PCR, is also suitable for real-time PCR. In the case of mammalian genomic DNA,  1~10 ng genomic DNA is sufficient for real-time PCR. 

    PROTOCOL:
    -This premix should be fully thawed at room temperature in the bags, gently vortexed and briefly centrifuged.
    Notes: Due to the high concentration stabilizer，there may be crystal precipitation in the premix ，which can be used normally after being fully thawed at room temperature .If used within 6 months,it should be stored at 2-8°C in the dark bag.
    -Purified DNA and RT reactions can be may be used directly or after dilution.
    -In order to reducing the artificial error of sampling， design the plate layout and sampling method by the number of the templates and primer pairs. According to the following two situations，the total reaction is divided into two parts for premixing and loading in the a thin-walled qPCR tube or plate at room temperature.

           Fore more genes and less samples in one plate
    Components   20μL reaction ×n   Operation
    qPCR Master Mix                  10μL×n Premix and Loading
    Template DNA Dilutions                           2μL×n
    2μM Forward primer                                4μL×n Premix and Loading
    2μM Reverse primer                                4μL×n

    Fore more samples and less genes in one plate
    Components   20μL reaction ×n  Operation
    qPCR Master Mix                    10μL×n Premix and Loading
    8μM Reverse primer      1μL×n Premix
    8μM Reverse primer      1μL×n
    Template DNA Dilutions                             8μL×n Premix and Loading







    -Gently mix the reaction solutions and spin down in microcentrifuge.

    CYCLING CONDITIONS:
     
    2-step PCR protocol
    1 Pre-denaturation 95℃ 60 sec 1cycle
    2 Denaturation 95°C 10 sec 40
    cycles  
    Annealing/ Extension 60°C 30 sec
    Data collection should be performed at the extension step.  

    3-step PCR protocol
    1 Pre-denaturation 95℃ 60 sec 1cycle
    2 Denaturation 95°C 15 sec 40
    cycles 
    Annealing 55-65°C 15 sec
    Extension 72°C 45 sec
    Data collection should be performed at the extension step.  

    Notes： 
    -Use this protocol first and optimize PCR conditions when necessary. Perform 3-step PCR when using primers with low Tm values or when 2-step PCR is not feasible. 
    -The annealing temperature can be set to 55~65°C, depending on the primer Tm value.
    -The annealing time should be set for 5~20 seconds. Longer annealing time results increased efficiency, and a shorter  time decreases non-specific amplification.
    -The pre-denaturation condition described above is sufficient  for inactivation of the anti-Taq DNA polymerase  antibodies   used in Hot Start PCR. To prevent unexpected  and inappropriate results, do not prolong the predenaturation period. 
    -Data collection step should be longer than 10 sec.
    -If commercially available primers are employed, the recommended conditions from each company should  be used. 

    DETECTION:
    -This reagent can be used in general detection devices,not needing ROX such as: LineGene(bioer);LightCycler (Roche);iCycler iQ,CFX96(Biorad/MJ); Thermal Cycler Dice(Takara);
    -This reagent with 1× ROX can also be used in detection equipment using passive reference, such as:ABI PRISM 7000, 7700, 7900 ,7300;Step One,Step one plus etc.(ABI) ,ABI PRISM 7500, 7500Fast(ABI); Mx3000P,3005P,MX4000,etc.(Agilent).

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Catalog Number

  • Amount:500reactions
  • Price:75.00$

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