Extraction of RNA from Agarose Gels using the Monarch® RNA Cleanup Kits

The Monarch RNA Cleanup Kits can be used to extract RNA from agarose gels, although this is not their primary application. Recoveries from extraction of RNA from agarose range from 40-70%, which is less than expected recovery for RNA Cleanup from enzymatic reactions.

Before You Begin:

  • Add 4 volumes of ethanol (≥ 95%) to the Monarch RNA Wash Buffer before use, as directed on the bottle

  • All centrifugation steps should be carried out at 16,000 x g. (~13K RPM in a typical microcentrifuge). This ensures all traces of buffer are eluted at each step.


  1. Excise the RNA fragment to be purified from the agarose gel using a razor blade, scalpel or other clean cutting tool. Use care to trim excess agarose from the perimeter of the band to minimize the amount of binding buffer needed, and reduce the time necessary to extract the RNA.

  2. Transfer the excised gel slice to a 1.5 ml microcentrifuge tube and weigh it. Using UV light to visualize the slice is common, but exposure time should be kept as short as possible to minimize damage to the RNA. Use long-wave UV when possible, as shorter wavelengths induce greater damage.

  3. Add 4 volumes of Monarch RNA Cleanup Binding Buffer to the tube with the slice. For example, add 400 μl to a 100 mg gel slice. Incubate the sample between 37–55°C (typically 50°C), gently mixing periodically until the gel slice is completely dissolved (generally 5–10 minutes). Failure to dissolve all the agarose will decrease the recovery yield due to incomplete extraction of the RNA and potential clogging of the column by particles of agarose.

  4. Add 1 volume of ethanol (≥ 95%) to the sample and mix well by pipetting up and down or flicking the tube. Do not vortex; vortexing may cause degradation of the RNA.

  5. Incubate the sample between 37–55°C (typically 50°C) for an additional 5 minutes to ensure the agarose remains completely dissolved and to avoid potential clogging of the column.

  6. Insert an RNA cleanup column into a collection tube, load the sample onto the column and close the cap. Spin for 1 minute, then discard flow-through. For diluted samples with a volume larger than 900 μl, load a portion of the sample, spin, and then repeat as necessary.

     To save time, spin for 30 seconds, instead of 1 minute.

  7. Re-insert the column into the collection tube. Add 500 μl RNA Cleanup Wash Buffer and spin for 1 minute. Discard the flow-through.

  8. Repeat wash (Step 7).

  9. Transfer the column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If in doubt, re-spin for 1 minute before placing into clean microfuge tube.

  10. Elute in nuclease-free water according to the table below. Incubate the column and elution for 5 minutes at 65°C prior to spinning. The eluted RNA can be used immediately or stored at -70°C.

    T2030 6–20 µl 5 minutes 1 minute
    T2040 20–100 µl 5 minutes 1 minute
    T2050** 50–100 µl 5 minutes 1 minute

    * When cleaning up large amounts of RNA (> 100 μg, NEB #T2050), some precipitation may occur following the addition of the Monarch RNA Cleanup Binding Buffer and ethanol to the sample (Steps 1 and 2). A pellet containing the RNA of interest may form on the side of the column following the first binding spin (Step 3). To maximize recovery of this RNA, a second elution is recommended.

    ** Yield may slightly increase if a larger volume is used, but the RNA will be less concentrated.

     To save time, spin for 30 seconds, instead of 1 minute.


Additional Resources: