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.