TITLE: Purification of RNA from a DNA/RNA Extract
AUTHORS: Roey Angel, Eva Petrova

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The following protocol is intended as a downstream application for our </span><a href="https://protocols.io/view/total-nucleic-acids-extraction-from-soil-yw9fxh6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Total Nucleic Acids Extraction from Soil</span></a><span> protocol. This protocol describes how to purify RNA from a DNA and RNA extract using </span><a href="https://www.thermofisher.com/order/catalog/product/AM2238?SID=srch-srp-AM2238#/AM2238?SID=srch-srp-AM2238" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">TURBO™ DNase</span></a><span> and </span><a href="https://www.thermofisher.com/order/catalog/product/K0841#/legacy=www.thermoscientificbio.com" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">GeneJET RNA Cleanup and Concentration Micro Kit</span></a><span>. This protocol is a simplified and condensed version of the full protocols provided by the manufacturers.</span></div></div>

[STEPS]
?. [DNA digestion]
Prepare the following mixture in a 1.5 ml tube: to  of TNA extract  ( to   of DNA). per up to Complete to   with RNase-free water
10 µl
42 µl
1 µg
3 µg
[TURBO DNase buffer 10x]
[RNaseOUT]
[0,1M DTT]
[Turbo DNase]
[DNA]
50 µl
?. [DNA digestion]
Incubate at  for .
37 °C
?. [RNA purification]
Add .
[Binding Buffer]
?. [RNA purification]
Add .
[absolute ethanol]
?. [RNA purification]
Transfer the mixture to the Gene JET RNA Purification Micro Column preassembled with a collection tube. Centrifuge the column for . Discard the flow-through. Place the GeneJET RNA Purification Micro Column back into the collection tube.
Centrifuge: 14000 34, 1 min
?. [RNA purification]
Add  (supplemented with ethanol) to the GeneJET RNA Purification Micro Column and centrifuge for . Discard the flow-through and place the purification column back into the collection tube.
[Wash Buffer 1 ]
Centrifuge: 14000 34, 1 min
?. [RNA purification]
Add  (supplemented with ethanol) to the GeneJET RNA Purification Micro Column and centrifuge for . Discard the flow-through and place the purification column back into the collection tube.
[Wash Buffer 2]
Centrifuge: 14000 34, 1 min
?. [RNA purification]
Repeat step 7.
?. [RNA purification]
Centrifuge the empty GeneJET RNA Purification Micro Column for an additional  to completely remove residual Wash Buffer.
Centrifuge: 14000 34, 2 min
This step is essential to avoid residual ethanol in the purified RNA solution. The presence of ethanol in the RNA sample may inhibit downstream enzymatic reactions.
?. [RNA purification]
Transfer the GeneJET RNA Purification Micro Column into a clean 1.5 ml Collection Tube tube.
?. [RNA purification]
Add  to  or nuclease-free water to the GeneJET RNA Purification Micro Column. Centrifuge for  to elute the RNA.
10 µl
[RNA storage solution]
Centrifuge: 14000 33, 1 min
?. [RNA purification]
Discard the purification column. Use the purified RNA immediately in downstream applications or store at  or  until use. Proceed to cDNA synthesis using superscript IV
-20 °C
-80 °C
For prolonged storage (more than 1 month), storage at   is recommended.
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