Strand Displacement Amplification (SDA) Protocol using WarmStart® Nt.BstNBI (NEB #R0725)


  1. Set up the following reaction at room temperature; if preferred, reactions can be set up on ice:

    Component 25 µl Reaction Final Conc.
    10X Isothermal Amplification Buffer 2.5 µl 1X (contains 2 mM MgSO4)
    dNTP Mix (10 mM)   1 µl 0.4 mM each
    F/Fbump/R/Rbump Primers (25 µM each) 0.5 µl 0.5 µM each
    WarmStart Nt.BstNBI (10,000 U/ml) 1 µl 0.4 U/ µl
    Bst 2.0 WarmStart® DNA Polymerase
    (8,000 U/ml)
    1 µl 0.32 U/ µl
    DNA Sample variable variable
    LAMP fluorescent dye (50X)   0.5 µl 1X
    Nuclease-free Water   to 25 µl  

  2. Incubate at 55°C for 60 minutes.

  3. If desired, heat inactivate 80°C for 20 minutes.


General Guidelines:

  1. A 50X primer mix can be prepared with all 4 primers: F, Fbump, R and Rbump 25 µM each in TE or water.

  2. Reactions can be set up at room temperature when using both WarmStart Nt.BstNBI and Bst 2.0 WarmStart® DNA Polymerase.

  3. A no-template control should be included in the experiment to ensure amplification specificity.

  4. If optimization is desired, try titrating Mg2+ (2–10 mM final) or Bst 2.0 WarmStart DNA Polymerase (0.04–0.32 U/µl) or WarmStart Nt.BstNBI (0.05-0.4 U/µl), or changing reaction temperature (50–60°C).

  5. Forward and reverse primers must have the Nt.BstNBI site towards the 5´ end. NEB has adapted the following tail sequences from