Isothermal Reaction Guide - Alrad

Isothermal Reaction Guide

Isothermal Reaction Guide

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  • OptiGene recommends LAMP as the isothermal amplification method of choice and suggests the following LAMP reaction conditions to achieve optimal results:
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    (Final conc.)
    Isothermal Mastermix(1) 15ul
    FIP/BIP Primers 0.8 – 2.0μM
    F3/B3 Primers 0.2μM
    LoopF/LoopB Primers 0.4 – 1.0μM
    DNA or RNA(2) sample >10 copies
    Nuclease-free water to 25μl

    (1)ISO-001/ ISO-004 / ISO-001Tin Isothermal Mastermixes containing a fluorescent dsDNA intercalating dye. Components have been optimised for real-time fluorescence based detection. Additional MgSO4 and dNTPs are recommended for turbidometric or agarose gel detection. However, OptiGene strongly discourage opening the amplified reaction tube due to the extremely high risk of contamination. (2)The addition of 0.5U AMV-RT is recommended for optimum RT-LAMP reaction sensitivity.

     

  • Run reactions on a Genie® or a qPCR instrument for real-time fluorescence detection. [Amplification: 65°C for 20-30 minutes. Anneal: 98°C-80°C (0.01°C/sec)]
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    Alternatively:

    (Final conc.)
    10X Supplied iBuffer 1X
    GspSSD2.0 DNA Polymerase(1) 8U
    MgSO4 3-5mM(2)
    dNTP mix 0.4mM each(2)
    FIP/BIP Primers 0.8 – 2.0μM
    F3/B3 Primers 0.2μM
    LoopF/LoopB Primers 0.4 – 1.0μM
    DNA or RNA(3) sample >10 copies
    Betaine(4) 1M
    Nuclease-free water to 25μl

    (1) or Gsp, GspM3.0 or Tin(exo-) LF DNA Polymerase. (2) Concentrations have been optimised for real-time fluorescence SSDbased detection. Additional MgSO4 and dNTPs are required for turbidometric or agarose gel detection. However, OptiGene strongly discourage opening the amplified reaction tube due to the extremely high risk of contamination. (3)The addition of 0.5U AMV-RT is recommended for optimum RT-LAMP reaction sensitivity. (4)The addition of betaine is highly recommended to achieve consistent amplification and to improve the sensitivity and specificity of low copy detection. OptiGene highly recommend the addition of betaine, especially for RT-LAMP reactions.

     

  • Run reactions on a Genie® or a qPCR instrument in combination with a dsDNA intercalating fluorescent dye to enable real-time detection.
    [Amplification: 65°C for 20-30 minutes. Anneal: 98°C-80°C (0.01°C/sec)]
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