Home Resources General Guidelines for Monarch Spin High-Capacity RNA Cleanup Kit (3 mg)

General Guidelines for Monarch Spin High-Capacity RNA Cleanup Kit (3 mg)

  • The standard protocol for this kit provides an example case with 300 μl as starting sample volume, corresponding to approximately 3 mg of RNA synthesized from an in vitro transcription (IVT) reaction (for example, using the HiScribe® T7 High Yield RNA Synthesis Kit (NEB #E2040)) in a scaled reaction volume of 286 μl treated with 14 μl of DNase I-XT (NEB #M0570). Note that DNase I-XT treatment of scaled IVT reactions is recommended to limit total sample volume and maximize starting RNA concentration. For other sample volumes, up to a maximum of 800 μl and 3 mg of RNA per column, scale volumes of Monarch Buffer BX and ethanol accordingly.
  • Monarch Buffer WX is supplied as a concentrate. Follow buffer preparation guidelines outlined before use. 
  • There are two centrifugation speeds in this protocol: 10,000 x g for sample loading and 16,000 x g for wash and elution, carried out at room temperature. 
  • This protocol is not compatible with the use of a vacuum manifold, and all steps should be completed using centrifugation. 
  • When cleaning up large amounts of RNA (> 500 μg), precipitation will occur following the addition of the Monarch Buffer BX and ethanol to the sample (steps 1 and 2). Load the entire sample including all the precipitate onto the column. 
  • Avoid overloading the column when working with high RNA inputs. For RNA inputs >3 mg, divide the sample into aliquots containing a maximum of 3 mg RNA before adding Monarch Buffer BX and ethanol. If >3 mg RNA is processed as a single sample, addition of the Monarch Buffer BX and ethanol results in the formation of a large precipitate, making it difficult to divide the sample evenly across columns and increasing the risk of column overload and RNA loss. 
  • A pellet containing the RNA of interest may form on the side of the column following the first binding spin (step 3). 
  • If the RNA in the starting sample is > 3 mg (maximum capacity per column), and re-loading the column > 2 times, a pellet containing RNA of interest may form in the collection tube. The pellet in the collection tube can be resuspended with nuclease-free water and processed using the RNA cleanup protocol. 
  • The column has a maximum capacity of 800 μl. If the sample volume (after addition of Monarch Buffer BX and ethanol) exceeds 800 μl, load the sample in 600 μl increments to help ensure high purity in the eluate. Before proceeding to step 5, ensure that all sample has passed through the column during step 4. An additional spin may be used to remove any residual liquid.
  • Re-loading the column multiple times (> 5) during sample loading steps can make elution more challenging; additional elutions will ensure successful recovery.
  • For RNA size > 5 kb, it is recommended to use 500 μl nuclease-free water for the first elution. To maximize recovery, a second elution is necessary for RNA sizes ≥ 5 kb. 
  • After the first elution, a gel-like pellet containing RNA of interest may remain on the side of the column and membrane layer. Additional elution steps will maximize recovery.