Isothermal Reaction Guide
Please contact us (by pushing the 'Request a Quote' button above) and we will provide the correct contact point at OptiGene for the Primer Design Service
(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.
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.
[Amplification: 65°C for 20-30 minutes. Anneal: 98°C-80°C (0.01°C/sec)]