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  • ShortCut RNase III Digestion Protocol (M0245)

    Protocol

    1. Synthesize, quantify and purify the desired fragment of dsRNA to be digested. ShortCut RNAi Kit (NEB #E2450) and HiScribe Transcription Kit (NEB #E2000) provide reagents and protocols to synthesize long dsRNA.
    2. Combine the following, in order: 
      dH20: 70 µl – x µl 
      10X ShortCut Reaction Buffer: 10 µl 
      dsRNA: x µl (10 µg) 
      ShortCut RNase III: 10 µl 
      10X MnCl2: 10 µl 
      -----------------------------------------------------
      Total Volume: 100 µl
    3. Mix and incubate for 20 minutes at 37°C.
    4. Add 10 µl 10X EDTA to stop the reaction. Do not heat inactivate. Heat inactivation reduces the yield of siRNA.