TITLE: Quick Protocol for Monarch® Total RNA Miniprep Kit (NEB #T2010)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Quick Protocol for Monarch® Total RNA Miniprep Kit (</span><a href="https://www.neb.com/products/t2010-monarch-total-rna-miniprep-kit#Product%20Information" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">NEB #T2010</span></a><span>).</span></div><div class = "text-block">Quickly and easily purify up to 100 µg of high-quality total RNA from multiple sample types – all with one kit!</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">For use with blood, cells and tissues</li><li style = "counter-reset:ol0;list-style-type:disc;">Also works with tough to lyse samples (bacteria, yeast, plant)</li><li style = "counter-reset:ol0;list-style-type:disc;">Effectively purifies total RNA of all sizes, including small RNAs >20 nt</li><li style = "counter-reset:ol0;list-style-type:disc;">Efficient genomic DNA removal (column and DNase I-based)</li><li style = "counter-reset:ol0;list-style-type:disc;">Contains Proteinase K for processing of tissues and blood samples</li><li style = "counter-reset:ol0;list-style-type:disc;">Includes RNA Protection Reagent for sample preservation</li><li style = "counter-reset:ol0;list-style-type:disc;">Excellent value</li><li style = "counter-reset:ol0;list-style-type:disc;">Kit components available separately</li></ul></div></div>

[STEPS]
?. [PART 1: Sample Disruption and Homogenization]
Please select your starting material of the following:- Cultured Mammalian Cells- Mammalian Whole Blood (Fresh or Frozen)- Tissue or Leukocytes- Tough-to-Lyse Samples (bacteria, yeast, plant, etc.) using Mechanical Lysis
?. [PART 1: Sample Disruption and Homogenization]
Pellet cells by centrifugation (500 x g) for 1 min.
[Centrifugation]
?. [PART 1: Sample Disruption and Homogenization]
Discard supernatant.
?. [PART 1: Sample Disruption and Homogenization]
Resuspend pellet in RNA Lysis Buffer (according to table below) by pipetting gently to avoid foaming.   AB1AMOUNT OF CELLSVOLUME OF RNA LYSIS BUFFER2up to 3 x 106300 µl33 x 106&nbsp;to 1 x 107&nbsp;≥ 600 µl
AB1AMOUNT OF CELLSVOLUME OF RNA LYSIS BUFFER2up to 3 x 106300 µl33 x 106&nbsp;to 1 x 107&nbsp;≥ 600 µl
Do not place samples on ice. For frozen pellets, thaw briefly before resuspension.
?. [PART 2: RNA BINDING AND ELUTION]
Transfer up to 800 µl of the sample from PART 1 to a gDNA removal column  (light blue) fitted with a collection tube.For sample identification, label collection tubes, as gDNA removal columns will be discarded after spinning.
For sample volumes > 800 µl (column reservoir capacity), columns may be reloaded.
All centrifugation steps should be performed at 16,000 x g.
?. [PART 2: RNA BINDING AND ELUTION]
Spin for 30 seconds to remove most of the gDNA. SAVE THE FLOW-THROUGH (RNA partitions here). Discard the gDNA removal column.
[Spinning]
?. [PART 2: RNA BINDING AND ELUTION]
Add an equal volume of ethanol (≥ 95%) to the flow-through and mix thoroughly by pipetting. Do not vortex. To exclude RNA ≷ 200 nt, add only 1/2 volume ethanol to flow-through.
?. [PART 2: RNA BINDING AND ELUTION]
Transfer mixture to an RNA purification column  (dark blue) fitted with a collection tube. Spin for 30 seconds. Discard flow-through. If further gDNA removal is essential for downstream applications, proceed to on-column DNase I treatment, steps 9-11 (recommended). If not, proceed to Step 12.
[Spinning]
?. [PART 2: RNA BINDING AND ELUTION]
[Optional (but recommended)] On-column DNase I treatment for enzymatic removal of residual gDNA:Add 500 µl RNA Wash Buffer and spin for 30 seconds. Discard flow-through.
[RNA Wash Buffer]
[Spinning]
?. [PART 2: RNA BINDING AND ELUTION]
[Optional (but recommended)] In an RNase-free microfuge tube (not included), combine 5 µl DNase I with 75 µl DNase I Reaction Buffer and pipet directly to the top of the column matrix.
[DNase I]
[DNase I Reaction Buffer]
?. [PART 2: RNA BINDING AND ELUTION]
[Optional (but recommended)] Incubate for 15 minutes at room temperature.
[Incubation]
?. [PART 2: RNA BINDING AND ELUTION]
Add 500 µl RNA Priming Buffer and spin for 30 seconds. Discard flow-through.
[RNA Priming Buffer]
[Spinning]
?. [PART 2: RNA BINDING AND ELUTION]
Add 500 µl RNA Wash Buffer and spin for 30 seconds. Discard flow-through.
[RNA Wash Buffer]
[Spinning]
?. [PART 2: RNA BINDING AND ELUTION]
Add another 500 µl RNA Wash Buffer and spin for 2 MINUTES.
[RNA Wash Buffer]
[Spinning]
?. [PART 2: RNA BINDING AND ELUTION]
Transfer column to an RNase-free microfuge tube (not included). Use care to ensure the tip of the column does not contact the flow-through. If in doubt, re-spin for 1 minute to ensure no ethanol is carried over.
[Re-spinning]
?. [PART 2: RNA BINDING AND ELUTION]
Add 30-100 µl Nuclease-free Water directly to the center of column matrix and spin for 30 seconds.
[Spinning]
For best results, elute with at least 50 µl, which is the minimum volume needed to wet the membrane.  Lower volumes can be used but will result in lower recovery (elution in 30 µl results in > 80% recovery and 100 µl provides maximum recovery).  For spectrophotometric analysis of eluted RNA, it may be necessary to re-spin eluted samples and pipet aliquot from top of the liquid to ensure that the A 260/230 is unaffected by possible elution of silica particles.
?. [PART 2: RNA BINDING AND ELUTION]
Place RNA on ice if being used for downstream steps, at -20°C for short-term storage (less than 1 week), or at -80°C for long-term storage. Addition of EDTA to 0.1-1.0 mM may reduce the activity of any contaminating RNases.
?. [PART 1: Sample Disruption and Homogenization]
Add an equal volume (up to 200 µl) of DNA/RNA Protection Reagent (2X concentrate) to an aliquot of whole blood and vortex briefly.
Do not place samples on ice. For frozen samples, quickly thaw in the presence of 2X DNA/RNA Protection Reagent while vortexing or shaking.
?. [PART 1: Sample Disruption and Homogenization]
For every 400 µl of DNA/RNA Protection Reagent/blood mixture, add 10 µl of Prot K. Vortex briefly and incubate at room temperature for 30 min.
[Incubation]
?. [PART 1: Sample Disruption and Homogenization]
Add an equal volume of isopropanol (not included) and vortex briefly.
?. [PART 2: RNA BINDING AND ELUTION]
Transfer mixture to an RNA purification column  (dark blue) fitted with a collection tube. Spin for 30 seconds. Discard flow-through. If further gDNA removal is essential for downstream applications, proceed to on-column DNase I treatment, steps 9-11 (recommended). If not, proceed to Step 9.
[Spinning]
?. [PART 2: RNA BINDING AND ELUTION]
[Optional (but recommended)] On-column DNase I treatment for enzymatic removal of residual gDNA:Add 500 µl RNA Wash Buffer and spin for 30 seconds. Discard flow-through.
[RNA Wash Buffer]
[Spinning]
?. [PART 2: RNA BINDING AND ELUTION]
[Optional (but recommended)] In an RNase-free microfuge tube (not included), combine 5 µl DNase I with 75 µl DNase I Reaction Buffer and pipet directly to the top of the column matrix.
[DNase I]
[DNase I Reaction Buffer]
?. [PART 2: RNA BINDING AND ELUTION]
[Optional (but recommended)] Incubate for 15 minutes at room temperature.
[Incubation]
?. [PART 2: RNA BINDING AND ELUTION]
Add 500 µl RNA Priming Buffer and spin for 30 seconds. Discard flow-through.
[RNA Priming Buffer]
[Spinning]
?. [PART 2: RNA BINDING AND ELUTION]
Add another 500 µl RNA Wash Buffer and spin for 2 MINUTES.
[RNA Wash Buffer]
[Spinning]
?. [PART 2: RNA BINDING AND ELUTION]
Transfer column to an RNase-free microfuge tube (not included). Use care to ensure the tip of the column does not contact the flow-through. If in doubt, re-spin for 1 minute to ensure no ethanol is carried over.
[Re-spinning]
?. [PART 2: RNA BINDING AND ELUTION]
Add 30-100 µl Nuclease-free Water directly to the center of column matrix and spin for 30 seconds.
[Spinning]
For best results, elute with at least 50 µl, which is the minimum volume needed to wet the membrane.  Lower volumes can be used but will result in lower recovery (elution in 30 µl results in > 80% recovery and 100 µl provides maximum recovery).  For spectrophotometric analysis of eluted RNA, it may be necessary to re-spin eluted samples and pipet aliquot from top of the liquid to ensure that the A 260/230 is unaffected by possible elution of silica particles.
?. [PART 2: RNA BINDING AND ELUTION]
Place RNA on ice if being used for downstream steps, at -20°C for short-term storage (less than 1 week), or at -80°C for long-term storage. Addition of EDTA to 0.1-1.0 mM may reduce the activity of any contaminating RNases.
?. [PART 1: Sample Disruption and Homogenization]
Determine the amount of 1X DNA/RNA Protection Reagent that you will need according to the table below. Prepare the 1X aliquot by diluting the 2X stock with nuclease-free water (not included for this step).  AB1Sample Input AmountVolume 1X DNA/RNA Protection Reagent2Tissue (up to 10 mg)300 µl3Tissue (10–30 mg)300–600 µl4Tissue (30–50 mg)≥ 600 µl5Leukocytes (up to 3 x 106)300 µl6Leukocytes (3 x 106&nbsp;to 1 x 107)≥ 600 µl
AB1Sample Input AmountVolume 1X DNA/RNA Protection Reagent2Tissue (up to 10 mg)300 µl3Tissue (10–30 mg)300–600 µl4Tissue (30–50 mg)≥ 600 µl5Leukocytes (up to 3 x 106)300 µl6Leukocytes (3 x 106&nbsp;to 1 x 107)≥ 600 µl
?. [PART 1: Sample Disruption and Homogenization]
Add 1X DNA/RNA Protection Reagent to sample.
Solid tissue samples should be submerged in protection reagent, not to exceed 10% (w/v). For maximal RNA recovery, tissues can be mechanically homogenized using a bead mill or similar device.
?. [PART 1: Sample Disruption and Homogenization]
For every 300 µl of DNA/RNA Protection Reagent/sample mixture, add 30 µl Prot K Reaction Buffer + 15 µl Prot K*. Vortex briefly and incubate according to the chart below:  AB1Sample TypeIncubation Time at 55°C**2Homogenized Tissues5 min3Solid Tissues5-30 min4Leukocytes30 min* Doubling the Prot K volume may increase RNA yield for some tissues.
** Longer incubation times may result in decreased RNA integrity. Optimal time should be determined empirically, as yield and integrity vary with incubation time.
AB1Sample TypeIncubation Time at 55°C**2Homogenized Tissues5 min3Solid Tissues5-30 min4Leukocytes30 min
[Homogenized Tissues / Solid Tissues]
[Leukocytes]
?. [PART 1: Sample Disruption and Homogenization]
Vortex sample briefly and spin for 2 min (16,000 x g) to pellet debris.
[Spinning]
?. [PART 1: Sample Disruption and Homogenization]
Transfer supernatant to an RNase-free microfuge tube (not included).
?. [PART 1: Sample Disruption and Homogenization]
Add an equal volume of RNA Lysis Buffer and vortex briefly.
?. [PART 2: RNA BINDING AND ELUTION]
Transfer up to 800 µl of the sample from PART 1 to a gDNA removal column  (light blue) fitted with a collection tube.For sample identification, label collection tubes, as gDNA removal columns will be discarded after spinning.
For sample volumes > 800 µl (column reservoir capacity), columns may be reloaded.
All centrifugation steps should be performed at 16,000 x g.
?. [PART 2: RNA BINDING AND ELUTION]
Spin for 30 seconds to remove most of the gDNA. SAVE THE FLOW-THROUGH (RNA partitions here). Discard the gDNA removal column.
[Spinning]
?. [PART 2: RNA BINDING AND ELUTION]
Add an equal volume of ethanol (≥ 95%) to the flow-through and mix thoroughly by pipetting. Do not vortex. To exclude RNA ≷ 200 nt, add only 1/2 volume ethanol to flow-through.
?. [PART 2: RNA BINDING AND ELUTION]
Transfer mixture to an RNA purification column  (dark blue) fitted with a collection tube. Spin for 30 seconds. Discard flow-through. If further gDNA removal is essential for downstream applications, proceed to on-column DNase I treatment, steps 9-11 (recommended). If not, proceed to Step 15.
[Spinning]
?. [PART 2: RNA BINDING AND ELUTION]
[Optional (but recommended)] On-column DNase I treatment for enzymatic removal of residual gDNA:Add 500 µl RNA Wash Buffer and spin for 30 seconds. Discard flow-through.
[RNA Wash Buffer]
[Spinning]
?. [PART 2: RNA BINDING AND ELUTION]
[Optional (but recommended)] In an RNase-free microfuge tube (not included), combine 5 µl DNase I with 75 µl DNase I Reaction Buffer and pipet directly to the top of the column matrix.
[DNase I]
[DNase I Reaction Buffer]
?. [PART 2: RNA BINDING AND ELUTION]
[Optional (but recommended)] Incubate for 15 minutes at room temperature.
[Incubation]
?. [PART 2: RNA BINDING AND ELUTION]
Add 500 µl RNA Priming Buffer and spin for 30 seconds. Discard flow-through.
[RNA Priming Buffer]
[Spinning]
?. [PART 2: RNA BINDING AND ELUTION]
Add 500 µl RNA Wash Buffer and spin for 30 seconds. Discard flow-through.
[RNA Wash Buffer]
[Spinning]
?. [PART 2: RNA BINDING AND ELUTION]
Add another 500 µl RNA Wash Buffer and spin for 2 MINUTES.
[RNA Wash Buffer]
[Spinning]
?. [PART 2: RNA BINDING AND ELUTION]
Transfer column to an RNase-free microfuge tube (not included). Use care to ensure the tip of the column does not contact the flow-through. If in doubt, re-spin for 1 minute to ensure no ethanol is carried over.
[Re-spinning]
?. [PART 2: RNA BINDING AND ELUTION]
Add 30-100 µl Nuclease-free Water directly to the center of column matrix and spin for 30 seconds.
[Spinning]
For best results, elute with at least 50 µl, which is the minimum volume needed to wet the membrane.  Lower volumes can be used but will result in lower recovery (elution in 30 µl results in > 80% recovery and 100 µl provides maximum recovery).  For spectrophotometric analysis of eluted RNA, it may be necessary to re-spin eluted samples and pipet aliquot from top of the liquid to ensure that the A260/230 is unaffected by possible elution of silica particles.
?. [PART 2: RNA BINDING AND ELUTION]
Place RNA on ice if being used for downstream steps, at -20°C for short-term storage (less than 1 week), or at -80°C for long-term storage. Addition of EDTA to 0.1-1.0 mM may reduce the activity of any contaminating RNases.
?. [PART 1: Sample Disruption and Homogenization]
Determine the amount of 1X DNA/RNA Protection Reagent that you will need according to the table below. Prepare the 1X aliquot by diluting the 2X stock with nuclease-free water (not included for this step).  AB1Sample Input AmountVolume 1X DNA/RNA Protection Reagent2Bacteria&nbsp;YeastPlant&nbsp;3≤ 5x107≤ 5x106&nbsp;400 µl4≤ 5x107-1x109≤ 5x106-5x107≤ 100 mg800 µl
AB1Sample Input AmountVolume 1X DNA/RNA Protection Reagent2Bacteria&nbsp;YeastPlant&nbsp;3≤ 5x107≤ 5x106&nbsp;400 µl4≤ 5x107-1x109≤ 5x106-5x107≤ 100 mg800 µl
?. [PART 1: Sample Disruption and Homogenization]
Add 1X DNA/RNA Protection Reagent to sample.
?. [PART 1: Sample Disruption and Homogenization]
Disrupt sample mechanically using a bead mill or similar device.
?. [PART 1: Sample Disruption and Homogenization]
Transfer lysate/homogenate to an RNase-free microfuge tube (not included).
?. [PART 1: Sample Disruption and Homogenization]
Spin for 2 min (16,000 x g) to pellet debris.
[Spinning]
?. [PART 1: Sample Disruption and Homogenization]
Transfer supernatant to an RNase-free microfuge tube (not included).
?. [PART 1: Sample Disruption and Homogenization]
Add an equal volume of RNA Lysis Buffer and vortex briefly.
?. [PART 2: RNA BINDING AND ELUTION]
Transfer up to 800 µl of the sample from PART 1 to a gDNA removal column  (light blue) fitted with a collection tube.For sample identification, label collection tubes, as gDNA removal columns will be discarded after spinning.
For sample volumes > 800 µl (column reservoir capacity), columns may be reloaded.
All centrifugation steps should be performed at 16,000 x g.
?. [PART 2: RNA BINDING AND ELUTION]
Spin for 30 seconds to remove most of the gDNA. SAVE THE FLOW-THROUGH (RNA partitions here). Discard the gDNA removal column.
[Spinning]
?. [PART 2: RNA BINDING AND ELUTION]
Add an equal volume of ethanol (≥ 95%) to the flow-through and mix thoroughly by pipetting. Do not vortex. To exclude RNA ≷ 200 nt, add only 1/2 volume ethanol to flow-through.
?. [PART 2: RNA BINDING AND ELUTION]
Transfer mixture to an RNA purification column  (dark blue) fitted with a collection tube. Spin for 30 seconds. Discard flow-through. If further gDNA removal is essential for downstream applications, proceed to on-column DNase I treatment, steps 9-11 (recommended). If not, proceed to Step 16.
[Spinning]
?. [PART 2: RNA BINDING AND ELUTION]
[Optional (but recommended)] On-column DNase I treatment for enzymatic removal of residual gDNA:Add 500 µl RNA Wash Buffer and spin for 30 seconds. Discard flow-through.
[RNA Wash Buffer]
[Spinning]
?. [PART 2: RNA BINDING AND ELUTION]
[Optional (but recommended)] In an RNase-free microfuge tube (not included), combine 5 µl DNase I with 75 µl DNase I Reaction Buffer and pipet directly to the top of the column matrix.
[DNase I]
[DNase I Reaction Buffer]
?. [PART 2: RNA BINDING AND ELUTION]
[Optional (but recommended)] Incubate for 15 minutes at room temperature.
[Incubation]
?. [PART 2: RNA BINDING AND ELUTION]
Add 500 µl RNA Priming Buffer and spin for 30 seconds. Discard flow-through.
[RNA Priming Buffer]
[Spinning]
?. [PART 2: RNA BINDING AND ELUTION]
Add another 500 µl RNA Wash Buffer and spin for 2 MINUTES.
[RNA Wash Buffer]
[Spinning]
?. [PART 2: RNA BINDING AND ELUTION]
Transfer column to an RNase-free microfuge tube (not included). Use care to ensure the tip of the column does not contact the flow-through. If in doubt, re-spin for 1 minute to ensure no ethanol is carried over.
[Re-spinning]
?. [PART 2: RNA BINDING AND ELUTION]
Add 30-100 µl Nuclease-free Water directly to the center of column matrix and spin for 30 seconds.
[Spinning]
For best results, elute with at least 50 µl, which is the minimum volume needed to wet the membrane.  Lower volumes can be used but will result in lower recovery (elution in 30 µl results in > 80% recovery and 100 µl provides maximum recovery).  For spectrophotometric analysis of eluted RNA, it may be necessary to re-spin eluted samples and pipet aliquot from top of the liquid to ensure that the A260/230 is unaffected by possible elution of silica particles.
?. [PART 2: RNA BINDING AND ELUTION]
Place RNA on ice if being used for downstream steps, at -20°C for short-term storage (less than 1 week), or at -80°C for long-term storage. Addition of EDTA to 0.1-1.0 mM may reduce the activity of any contaminating RNases.