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  • Ligation of 3´ and 5´ Adaptors (E6120)

    Overview

    Starting Material:
    1–10 μg total RNA. Alternatively, previously isolated small RNA from 1–10 μg total RNA can be used as starting material.

    Protocol

    1. Mix the following components in a sterile PCR tube:
      Input RNA: 1–6 μl
      3´ SR Adaptor 1: 1 μl
      Nuclease-free Water: variable
      ------------------------------------------------------------
      Total volume: 7 μl
    2. Incubate in a preheated thermal cycler for 2 minutes at 70°C.
    3. Transfer tube to ice.
    4. Add the following components:
      3´ Ligation Reaction Buffer (2X): 10 μl
      3´ Ligation Enzyme Mix: 3 μl 
      ------------------------------------------------------------
      Total volume: 20 μl
    5. Incubate for 1 hour at 25°C in a thermal cycler.
    6. Add the following components to the ligation mixture from step 5 and mix well:
      Nuclease-free Water: 4.5 μl
      SR RT Primer 1: 1 μl
      ------------------------------------------------------------
      Total volume now should be: 25.5 μl
    7. Heat samples for 5 minutes at 75°C. Transfer to 37°C for 30 minutes and then, to 25°C for 15 minutes.
    8. With 5 minutes remaining, resuspend the 5´ SR Adaptor 1 in Nuclease-free Water (For NEB #E6120S resuspend NEB #E6125A in 60 μl Nuclease-free Water and for NEB #E6120L resuspend NEB #E6125AA in 300 μl Nuclease-free Water).
    9. Heat the adaptor at 70°C for 2 minutes.
    10. Transfer tube to ice.
    11. Add the following components to the ligation mixture from step 7 and mix well:
      5´ SR Adaptor 1 (from Step 10): 1 μl
      5´ Ligation Reaction Buffer (10X): 1 μl
      5´ Ligation Enzyme Mix: 2.5 μl
      ------------------------------------------------------------
      Total volume now should be: 30 μl
    12. Incubate for 1 hour at 25°C in a thermal cycler.