How to Purify RNA using the Monarch Cleanup Kit
Monarch RNA Cleanup Kits can be used to purify RNA from in vitro transcription reactions or other enzymatic reactions, such as treat DNase I treatment.
We offer 3 products for RNA cleanup, each with a different binding capacity: 10 μg, 50 μg, and 500 μg. Each of these is available in 10 and 100 prep sizes. This protocol can be used for any of these kits.
Before Your Prep:
Before you begin, add 4 volumes of ethanol greater than or equal to 95% to the RNA Cleanup Wash Buffer concentrate. For the 10-prep kit, add 10 mls of ethanol. For the 100-prep kit, add 80 mls of ethanol per bottle.
Prepare Your Sample:
A starting sample volume of 50 μl is recommended. For smaller samples, nuclease-free water can be used to adjust the volume up to 50 ul. For samples larger than 50 μl, buffer volumes should be scaled accordingly.
Add Binding Buffer
Add 2 volumes of RNA Cleanup Binding Buffer, or 100 μl, to the 50 μl sample.
For cleanup of RNA greater than or equal to 25 nts, add 150 μl (or 1 volume) of ethanol. If you would like to cleanup RNA as small as 15 nucleotides, add 300 ul (or 2 volumes) of ethanol. Mix well by pipetting up and down or flicking the tube. Do not vortex.
Load Sample onto Column
Insert the column into a collection tube, load the sample onto the column and close the cap. Spin for 1 minute, then discard the flow-through. If your diluted sample exceeds 900 μl, you will need to load the sample onto the column, spin and then repeat as necessary, as the column can only hold 900 μl at a time.
Re-insert the column into the collection tube and add 500 μl of RNA Cleanup Wash Buffer, spin for 1 minute, and discard the flow-through.
Repeat Column Wash
Repeat the wash: add another 500 μl of RNA Cleanup Wash Buffer to the column, spin for 1 minute and discard the flow-through.
Prepare Column for Elution
Transfer the column to an RNase-free 1.5 ml microfuge tube. Be careful that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute to ensure traces of salt and ethanol are not carried over to the next step.
You are now ready to elute the RNA. Elution volumes and incubation times will differ for the 3 kits. Add the appropriate volume of nuclease-free water, as indicated in the table, to the center of the column matrix. For the 10 and 50 μg capacity columns, there is no need to incubate prior to the 1 minute elution spin.
Elute Sample RNA from Column
For the 500 μg capacity columns, incubate the column with nuclease-free water at room temp for 5 minutes prior to the 1 minute spin.. This elution can be repeated to increase yield. In all cases, yield may slightly increase if a larger volume is used, but the RNA will be less concentrated.
The eluted RNA can be used immediately or stored at -70°C. The Monarch RNA Cleanup Kits can also be used for RNA gel extraction and fractionation. Protocols can be found on the product web page and in the product manual.
Contact Technical Support
If you have questions about this protocol or any NEB product, you are always welcome to contact our scientists for technical support at [email protected]
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