ShortCut RNase III Digestion Protocol (M0245)


  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.