Home Protocols Protocol for ssDNA/RNA Ligation (NEB #M0319)

Protocol for ssDNA/RNA Ligation (NEB #M0319)


Protocol

  1. Set up the following reaction in a sterile microfuge tube:
    2. Components Volume
    ssDNA/RNA Substrate 20 pmol (1 pmol/ul) 
    5´ App DNA Oligonucleotide 200 pmol (10 pmol/µl)
    10X NEBuffer 1 2 µl
    50 mM MnCl2 (for ssDNA ligation only) 2 µl
    Thermostable 5´ App DNA/RNA Ligase 2 µl (40 pmol)
    Nuclease-free Water to 20 µl
  2. Incubate at 65°C for 1 hour. For DNA-DNA Ligations the ssDNA to 5’ AppDNA ligation occurs with reduced efficiency, therefore in these cases we recommend extending the incubation time overnight.
  3. We recommend using a thermocycler or heated top for reaction incubation to reduce evaporation.
  4. For inactivation of the enzyme we recommend Proteinase K treatment or heat-kill at 90°C for 3 minutes.
  5. Proteinase K treatment is recommended to release RNA bound by the ligase; or you may see a smear on a gel.