| Component | Final Concentration | Note |
|---|---|---|
| Isothermal Mastermix (1) | 15ul | 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. |
| 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 | The addition of 0.5U AMV-RT is recommended for optimum RT-LAMP reaction sensitivity. |
| Nuclease-free water | to 25μl |
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)
Alternatively:
| Component | Final Concentration | Note |
|---|---|---|
| 10X Supplied iBuffer | 1X | |
| GspSSD2.0 DNA Polymerase (1) | 8U | or Gsp, GspM3.0 or Tin(exo-) LF DNA Polymerase |
| MgSO4 (2) | 3-5mM | 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. |
| dNTP mix (2) | 0.4mM each | |
| 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 | The addition of 0.5U AMV-RT is recommended for optimum RT-LAMP reaction sensitivity. |
| Betaine (4) | 1M | 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. |
| Nuclease-free water | to 25μl |
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)
