TITLE: NEB Monarch® RNA Cleanup Kit
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is for the Monarch RNA Cleanup Kits (NEB #’s </span><a href="https://international.neb.com/products/t2030-monarch-rna-cleanup-kit-10-ug" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">#T2030</span></a><span>, </span><a href="https://international.neb.com/products/t2040-monarch-rna-cleanup-kit-50-ug" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">T2040</span></a><span>, and </span><a href="https://international.neb.com/products/t2050-monarch-rna-cleanup-kit-500-ug" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">T2050</span></a><span>). The protocol can also be used with the Monarch RNA Cleanup Columns (NEB #’s </span><a href="https://international.neb.com/products/t2037-monarch-rna-cleanup-columns-10-ug" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">T2037</span></a><span style = ":;:;">,</span><a href="https://international.neb.com/products/t2047-monarch-rna-cleanup-columns-50-ug" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">T2047</span></a><span>, and</span><a href="https://international.neb.com/products/t2057-monarch-rna-cleanup-columns-500-ug" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">T2057</span></a><span>) and associated buffers (NEB #’s </span><a href="https://international.neb.com/products/t2041-monarch-rna-cleanup-binding-buffer" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">T2041</span></a><span style = ":;:;">,</span><a href="https://international.neb.com/products/t2042-monarch-rna-cleanup-wash-buffer" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">T2042</span></a><span>).</span></div><div class = "text-block">The standard protocol outlined below will purify RNA ≥ 25 nt. A simple modification in Step 2 can allow for the purification of RNA as small as 15 nt.</div></div>

[STEPS]
?. Add  to the 50 μl sample. A starting sample volume of 50 μl is recommended. For smaller samples, nuclease-free water can be used to adjust the volume. For samples larger than 50 μl, scale buffer volumes accordingly. Samples with a starting volume > 150 μl will require reloading of the column during Step 3.
[RNA Cleanup Binding Buffer]
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 (Step 7) is recommended.
?. Add  to your sample and mix by pipetting or flicking the tube. Do not vortex. This will enable the binding of RNA ≥ 25 nt. If you wish to bind RNA as small as 15 nt, add 2 volumes (300 μl) of ethanol to your sample instead of 1 volume (150 μl). The addition of 2 volumes of ethanol shifts the cutoff size of RNA binding from 25 nt down to 15 nt.
[ethanol (≥95%)]
?. Repeat wash: Re-insert column into collection tube. Add  and spin for  at  . Discard the flow-through.
[RNA Cleanup Wash Buffer]
Centrifuge: 16000 33
To save time, spin for 30 seconds, instead of 1 minute.
?. Transfer column to an RNase-free 1.5 ml microfuge tube (not provided). Use care to ensure 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 next step.
?. 9 B
?. 9 A
?. [Section ONE ]
10 A
?. [Section ONE ]
9 B
?. 9 A
?. 10 A
?. Insert column into collection tube, load sample onto column and close the cap. Spin for  at  , then discard flow-through. For diluted samples > 900 μl, load a portion of the sample, spin, and then repeat as necessary.
Centrifuge: 16000 33
To save time, spin for 30 seconds, instead of 1 minute.
?. Re-insert column into collection tube. Add  and spin for  at  . Discard the flow-through.
[RNA Cleanup Wash Buffer]
Centrifuge: 16000 33
To save time, spin for 30 seconds, instead of 1 minute.
?. Elute in nuclease-free water at  according to the table below. The eluted RNA can be used immediately or stored at -70°C. Care should be used to ensure the elution buffer is delivered onto the matrix and not the wall of the column to maximize elution efficiency.  ABCD1KitElution VolumeIncubation TimeSpin Time2T20306-20 µlN/A1 minute3T204020-100 µlN/A1 minute4T2050**50-100 µl5 minutes (room temp.)1 minute
Centrifuge: 16000 33
ABCD1KitElution VolumeIncubation TimeSpin Time2T20306-20 µlN/A1 minute3T204020-100 µlN/A1 minute4T2050**50-100 µl5 minutes (room temp.)1 minute
To save time, spin for 30 seconds, instead of 1 minute.