The following protocol is for an On-Membrane DNase I digestion (Note: This DNase I set is formulated for on column use ONLY). Prepare all materials required before starting the RNA isolation procedure to minimize RNA degradation. Follow the E.Z.N.A® RNA protocol until the optional step for on-membrane DNase I digestion.

1. For each HiBind® RNA column, prepare the DNase I digestion reaction mixture as follows:

   OBI DNase I Digestion Buffer	73.5 µl
   RNase-free DNase I (20 Kunitz units/µl)	1.5 µl
   Total volume	75 µl

   
   Note:
   1. DNase I is very sensitive and is subject to physical denaturation, so do not vortex this DNase I mixture. Mix gently by inverting the tube. Prepare the fresh DNase I digestion mixture before beginning the RNA isolation procedure.
   2. OBI DNase I digestion buffer is supplied with OBI RNase-free DNase set.
   3. Standard DNase buffers may not be compatible with on-membrane DNase I digestion using the Omega Bio-Tek kit
   
    
2. Pipet 75 µl of the DNase I digestion reaction mixture directly onto the surface of the HiBind® RNA membrane in each column. Make sure to pipet the DNase I digestion mixture directly onto the membrane. DNase I digestion will not be complete if some of the mixture sticks to the wall or to the O-ring of the HiBind® RNA column.
    
3. Incubate at room temperature (25-30°C) for 15 minutes.
    
4. Wash the HiBind® RNA column with RNA Wash Buffer I & RNA Wash Buffer II by following the standard E.Z.N.A.® RNA protocol.