In some situations, such as if the RNA input is > 3 mg (maximum capacity per column) and/or the column is re-loaded > 2 times, a pellet containing RNA of interest may form in the collection tube. Any pellet formed in the collection tube can be resuspended by discarding the supernatant in the collection tube, resuspending the pellet with nuclease-free water and processing it through the RNA clean up protocol.
Avoid overloading the column when working with high RNA inputs. For RNA inputs > 3 mg, split the sample into aliquots containing a maximum of 3 mg RNA before adding Monarch Buffer BX and ethanol. Once Buffer BX and ethanol are added, a large precipitate forms, making equal column loading difficult, thereby increasing the risk of column overload and potential RNA loss.
