Strand Displacement Amplification (SDA) Protocol using WarmStart® Nt.BstNBI (NEB #R0725)
Procedure:
- 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
- Incubate at 55°C for 60 minutes.
- If desired, heat inactivate 80°C for 20 minutes.
General Guidelines:
- A 50X primer mix can be prepared with all 4 primers: F, Fbump, R and Rbump 25 µM each in TE or water.
- Reactions can be set up at room temperature when using both WarmStart Nt.BstNBI and Bst 2.0 WarmStart® DNA Polymerase.
- A no-template control should be included in the experiment to ensure amplification specificity.
- 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).
- Forward and reverse primers must have the Nt.BstNBI site towards the 5´ end. NEB has adapted the following tail sequences from https://pubmed.ncbi.nlm.nih.gov/15975661/):
Forward: 5′ ACCGCATCGAATGCATGTGAGTCAAAA–target
Reverse: 5′ GGATTCCGCTCCAGACTTGAGTCAAAA–target