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  • Ligation of 3´ and 5´ Adaptors (E6160)

    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. For total RNA input of < 5 μg, dilute the 3´ SR Adaptor 3 1:2 in Nuclease-free Water.

      Mix the following components in a sterile PCR tube:
      Input RNA: 1–6 μl
      NEBNext 3´ SR Adaptor 3: 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 (For total RNA input of < 5 μg, dilute the SR RT Primer 3 1:2 in Nuclease-free Water):
      Nuclease-free Water: 4.5 μl
      SR RT Primer 3: 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 3 in Nuclease-free Water (For NEB #E6160S resuspend NEB #E6162A in 30 μl Nuclease-free Water and for NEB #E6160L resuspend NEB #E6162AA in 150 μl Nuclease-free Water).

      For total RNA input of < 5 μg, resuspend the 5´ SR Adaptor 3 NEB #E6162A in 60 μl Nuclease-free Water and NEB #E6162AA 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 3 (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.