TITLE: Total Nucleic Acids Extraction from Soil
AUTHORS: Roey Angel, Eva Petrova, Ana Lara-Rodriguez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The following protocol is intended for the simultaneous extraction of DNA and RNA (total nucleic acids, or TNA) from various soil and sediment samples. The protocol was designed based on two protocols published by </span><a href="http://10.1128/AEM.65.5.1980-1990.1999" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Henckel et al. (1999)</span></a><span> and </span><a href="http://10.1128/AEM.66.12.5488-5491.2000" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Griffiths et al. (2000)</span></a><span>, with several critical modifications. Recently, we have added the option to include an ammonium aluminium sulfate salt for soils with high humic content (</span><a href="http://10.1016/S0167-7012(02)00210-5" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Braid et al., 2003</span></a><span>). The result is a highly flexible and streamlined protocol, which delivers high yields of nucleic acids with quality suitable for all downstream molecular applications from most types of soil and sediment samples. Please cite </span><a href="http://10.1038/ismej.2011.141" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Angel et al. (2012)</span></a><span style = ":;">.</span></div><br/><br/><br/><br/><br/><br/><br/></div>

[STEPS]
?. [Solutions for TNA extraction]
Prepare the following solutions for TNA extractionUse clean and preferably baked glassware (make sure all non-glass components can withstand the high temperatures).
?. [Solutions for TNA extraction]
One of the following phosphate buffers:Phosphate buffer  ( ,   )Dissolve the salts in RNase-free water and fill up to 500 ml. Autoclave.Phosphate buffer  ( ,   )Dissolve the salts in RNase-free water and fill up to 500 ml. Autoclave.Phosphate buffer + AlNH4 (120 mM PB pH 8.0 + 0.1M AlNH4(SO4)2•12H2O)Dissolve in PB. Autoclave.Store at
[K2HPO4•3 H2O (M.W. 228.22)]
[KH2PO4 (M.W. 136.09) ]
[RNase-free water]
[K2HPO4•3 H2O  (M.W. 136.09) ]
[KH2PO4  (M.W. 136.09) ]
[RNase-free water]
[ AlNH4(SO4)2 · 12H2O (M.W. 453.33)]
[120 mM PB pH 8.0]
0 Room temperature
?. [Solutions for TNA extraction]
Either TNC or TNSTNCDissolve the salts in RNase-free water and fill up to 200 ml. Autoclave.TNSDissolve the salts in RNase-free water and fill up to 200 ml. Autoclave.Store at
[TRIZMA (M.W. 121.14) ]
[NaCl]
[CTAB (M.W 364.45)]
[RNase-free water]
[TRIZMA (M.W 121.14)]
[NaCl]
[SDS (M.W 288.38) ]
[RNase-free water]
0 Room temperature
?. [Solutions for TNA extraction]
PEG precipitation solution (30%)Add ingredients to a graduated Duran bottle. Add water to fill up to 200 ml, shake vigorously by hand, autoclave and mix well while hot (solution turns milky when hot, but then turns clear when cooled to room temperature).Store at
[PEG (M.W 7000-9000)]
[NaCl]
[RNase-free water]
0 Room temperature
?. [Solutions for TNA extraction]
Molecular-grade ethanol solution (75%)Store at
[Absolute ethanol]
[RNase-free water]
-20 °C
?. [Solutions for TNA extraction]
Low-EDTA TE bufferPrepare in a laminar-flow hood (to protect stocks), filter sterilise (0.2 µm) and autoclave.Store at
[Tris-HCl 1 M, pH 8.0 (Trizma)]
[EDTA 0.5 M, pH 8.0]
[RNase-free water]
0 Room temperature
?. [Extraction]
Weigh   (0.2-0.7 g) into a Lysing Matrix E tube, or a 2-ml screw-cap tube filled with 0.7 g (one full PCR tube) of 0.1 mm glass beads and place the tube  .
[soil]
on ice
?. [Extraction]
Add  of either PB pH 8.0, PB pH 5.8 or PB+Al,  of TNC or TNS and  of TE-saturated phenol (if using PB pH 8.0) or water-saturated phenol (if using PB pH 5.8). Alternatively, add of PB,of TNC or TNS, if not using phenol (see Guidelines and Warnings on which combination of solutions to choose.)
375 µl
125 µl
400 µl
675 µl
225 µl
?. [Extraction]
Immediately place the tube in a sample homogeniser and process for  at 6.5 m s-1. We recommend using the FastPrep-24™ sample homogenizer with the CoolPrep™ adapter for 24 x 2 ml  . If using the CoolPrep™ adapter make sure all slots are filled with either sample or empty tubes.
[(dry ice)]
?. [Extraction]
Chill for  (not necessary if using the CoolPrep™ adapter).
on ice
?. [Extraction]
Centrifuge at  (centrifugation at RT is also possible).
Centrifuge: 14000 33, 3 min, 15 10
?. [Extraction]
Transfer the entire liquid (aqueous and organic phases) to a fresh 2 ml tube by decanting or pipetting. tubes.
Your supernatant has two phases. An aqueous upper phase that should be transparent/translucent and a phenol phase that should be opaque. Make sure to pour both into the fresh 2 ml tube.
?. [Extraction]
Repeat Steps 3-7 two more times using the same Lysing Matrix E tube. Be careful not to overfill the tube as this might cause phenol leakage during the sample homogenising process. If there is not enough space in the tube for all the reagents, decrease the phenol volume (down to 200 μl). For the second homogenising repetition use a fresh 2 ml tube to collect the supernatant in Step 7, while for the third repetition divide the supernatant between the two tubes from the previous repetitions to achieve equal volumes.
?. [Extraction]
Add   (or 1 volume) to each of the tubes containing the supernatant, to a maximum of 2 ml total volume in each tube.
[phenol/chloroform/isoamyl alcohol 25:24:1]
?. [Extraction]
Mix the two phases , by hand or using a vortex. Centrifuge  .
Centrifuge: 14000 33, 3 min, 15 10
?. [Extraction]
Using a 1-ml pipette tip, carefully transfer the aqueous phase (the upper phase) from each tube to two fresh 2 ml tubes.Be careful not to touch or pipette the interphase or the organic phase (the lower phase)
If, by mistake, some of the interphase or organic phase were pipetted, simply return the liquid to the original tube and centrifuge it again.
?. [Extraction]
Add   (or 1 volume) to each tube.
[chloroform/isoamyl alcohol 24:1]
?. [Extraction]
Mix the phases vigorously by hand or using a vortex. Centrifuge  .
Centrifuge: 14000 33, 3 min, 15 10
?. [Extraction]
Carefully transfer the supernatant from each tube to a fresh 2 ml low-binding microcentrifuge tubes. At this point, you should have two low-binding tubes per sample.
If, by mistake, some of the interphase or organic phase were pipetted, simply return the liquid to the original tube and centrifuge it again.
?. [Extraction]
Add to each tube  and  (or twice the extract’s volume).
[RNA-grade glycogen]
[PEG Precipitation Solution]
?. [Extraction]
Centrifuge  .
Centrifuge: 14000 33, 60 min, 4 10
?. [Extraction]
Decant the supernatant, briefly centrifuge or spin-down again to collect the drops and using a pipette remove as much as possible from the remaining precipitation solution. Be careful not to disturb the pellet.
?. [Extraction]
Add , invert the tube several times. Centrifuge at .
[ice-cold 75% EtOH]
Centrifuge: 14000 33, 20 min, 4 10
?. [Extraction]
Remove the supernatant using a pipette, shortly centrifuge again to collect the drops and using a pipette remove as much as possible from the remaining ethanol. Be careful not to disturb the pellet.
?. [Extraction]
Leave the tubes open at  for approximately  (preferably under a flame, inside a laminar-flow hood, or inverted on a piece of kitchen paper, to prevent contamination) to evaporate the remaining ethanol. Alternatively, the pellets can be dried under a filtered stream of N2, or in a speed-vac.
0 Room temperature
The pellets might not be completely dry at this point, but the remaining liquid should be pure water.
?. [Extraction]
Resuspend each pellet in  to   and combine both subsamples into one of the non-stick tubes.
50 µl
[Low-EDTA TE Buffer]
?. [Purification]
Remove co-extracted humic substances using OneStep™ PCR Inhibitor Removal KitThis step is optional but very recommended.
?. [Purification]
Resuspend the resin in the column by brief vortexing (if the column is dry, add 100 μl of RNAse-free water prior to vortexing.)
?. [Purification]
Loosen the cap by a quarter twist, place the column in a collection tube and centrifuge
Centrifuge: 8000 34, 3 min
?. [Purification]
Place the column in a fresh 1.5 ml tube, pipette the entire TNA extract (up to 200 μl) on top of the resin.
Be careful not to disturb the resin with the tip
?. [Purification]
Centrifuge
Centrifuge: 8000 34, 1 min
?. [Purification]
Discard the column and retain the extract in the tube.
?. [Quantification]
Optional: load   to of the total NA extract on an agarose gel to evaluate its quantity and quality using electrophoresis.
5 µl
10 µl
?. [Quantification]
Quantify total NA extract using RiboGreen™ and PicoGreen™ (see Guidelines and Warnings) or only DNA using PicoGreen™.
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?. [Storage]
The extract can be used directly as a DNA template for downstream applications. We recommend aliquoting  to  as a work-template to be stored at  , in order to minimise freeze-thaw cycles.
10 µl
100 µl
-20 °C
?. [Storage]
Depending on quality and quantity, aliquot  to  for RNA purification. Proceed to Purification of RNA from a DNA/RNA Extract.
10 µl
100 µl
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?. [Storage]
If the removal of RNA is necessary, follow up with RNaseH treatment and purification using e.g. Genomic DNA Clean & Concentrator.