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[DESCRIPTION]
<p><span style="font-weight: 400;">Mu-DNA is designed to be a simple and modular spin column-based DNA extraction ‘toolkit’ for the isolation of high purity DNA from multiple sample types. Here we present optimised Mu-DNA protocols for soil, tissue and water samples. Each protocol consists of five stages for DNA extraction: lysis, inhibitor removal, silica binding, wash and elution. Mu-DNA: Tissue has no inhibitor removal stage. All stages are designed to be modular and interchangeable between protocols. For example, an inhibitor removal stage can be added to a tissue protocol or a tissue protocol wash stage added to a soil protocol. The modular aspect allows for the creation of an optimised, bespoke DNA extraction for a specific sample type.</span></p>

[BEFORE_START]
<p><strong>Incubator</strong><br />Preheat incubating apparatus to 55°C.</p>
<p><strong>Samples</strong><br />Defrost samples (if necessary) at room temperature. Samples stored in ethanol and other storage buffers (e.g. RNAlater) are best air dried on sterile blotting paper (or similar) to remove as much storage buffer as possible prior to lysis.</p>
<p><strong>Working solutions</strong><br />Create the relevant working solutions in sufficient volumes required for the extraction method.</p>
<p><strong>Important:</strong> Incubate <em><strong>Soil Lysis Additive</strong></em>, <em><strong>Tissue Lysis additive</strong></em>, <em><strong>Water Lysis Additive</strong></em>, <em><strong>Binding Solution</strong></em> and <em><strong>Tissue Binding Solution</strong></em> at 55°C until required. If any precipitate is present mix gently until redissolved. Use solutions while still warm.</p>
<p><strong>Lysis master mix</strong><br />To reduce processing time when extracting from a large number of samples it is possible to create a master mix of reagents used in the lysis stage of each protocol (Soil: <em><strong>Lysis Solution</strong></em> and <em><strong>Soil Lysis Additive</strong></em>; Tissue: <em><strong>Lysis Solution</strong></em>, <em><strong>Tissue Lysis Additive</strong></em> and <em><strong>PK</strong></em>; Water: <em><strong>Lysis Solution</strong></em> and <em><strong>Water Lysis Additive</strong></em>). Heat the master mix at 55°C until required, mix gently occasionally. This prevents the formation of precipitates that interfere with lysis. Use the master mix while still warm.</p>

[GUIDELINES]
<p><strong>Materials required</strong><br />Below are listed the materials required for all Mu-DNA extraction protocols. Companies used and product codes are provided.</p>
<p><strong>Chemicals</strong></p>
<p>Guanidine thiocyanate (Alfa Aesar: B21250.22)<br />Trisodium phosphate dodecahydrate (Sigma Aldrich: 04277-1KG)<br />Sodium chloride (Sigma Aldrich: S7653-250G)<br />Tris HCl (Alfa Aesar: J67233.22)<br />Disodium EDTA dihydrate (Sigma Aldrich: E5134-250G)<br />Sodium dodecyl sulphate (Alfa Aesar: J75819.22)<br />Proteinase K (Thermofisher: AM2542)<br />Ammonium acetate (Sigma Aldrich: A1542-500G)<br />Aluminium ammonium sulphate dodecahydrate (Alfa Aesar: 13802.22)<br />Calcium chloride dihydrate (Sigma Aldrich: 1.02382.0250)<br />Guanidine hydrochloride (Thermofisher: 10071503)</p>
<p><strong>Plastics</strong></p>
<p>2 mL screw cap tubes (Starlab: E1420-2341)<br />7mL Bijou tubes (Sigma-Aldrich: 129A)*<br />1.5 mL tubes (Starlab: S1615-5510)<br />2 mL tubes (Starlab: E1420-2000)<br />Spin Columns (NBS Biologicals: SD5005)</p>
<p>* these tubes are the ones used in our study and fit in a TissueLyser II but do not fit in many centrifuges. Alternatively you can use transport tubes (Axygen SCT-5ML-S, Fisher Scientific: 12559107) that fit in a Mobio Vortex Adapter and all centrifuges with 15 mL falcon tube fittings.</p>
<p><strong>Garnet beads</strong></p>
<p>Garnet grit was sourced from an abrasives company at the grades required (0.15 mm and 1 - 1.4 mm). Each grade was thoroughly washed with purified water through a suitable mesh sieve to remove any detritus and fine particles. After washing, the garnet grit was transferred to 250 mL conical flasks capped with aluminium foil and sterilised at 210°C for three hours. The garnet grit was left to cool before being transferred to sterile 50 mL falcon tubes. Alternatively sterile garnet bead tubes, or similar, can be purchased commercially (e.g. Qiagen).</p>
<p><strong>Stock solutions</strong></p>
<p>Stock solutions are given as compositions for 100 mL with the exception of PK.</p>
<p><em><strong>1 M Tris HCl (pH 8)</strong></em><strong>:</strong><br />Dissolve 15.7 g of Tris HCl in 75 mL ddH<sub>2</sub>O. Adjust to pH 8 with 5 M NaOH. Bring to 100 mL with ddH<sub>2</sub>O.</p>
<p><em><strong>0.5 M EDTA (pH 8)</strong></em><strong>:</strong><br />Dissolve 18.6 g of disodium EDTA dihydrate in 75 mL ddH<sub>2</sub>O. Adjust to pH 8 with 5 M NaOH. Bring to 100 mL with ddH<sub>2</sub>O.</p>
<p><em><strong>20% SDS</strong></em><strong>:</strong><br />Dissolve 20 g sodium dodecyl sulphate in 75 mL ddH<sub>2</sub>O, bring to 100 mL with ddH<sub>2</sub>O.</p>
<p><em><strong>PK</strong></em><strong>:</strong><br />To 7 mL ddH<sub>2</sub>O add 0.5 mL 1 M Tris HCl (pH 8) and 100 mg Proteinase K, bring to 10 mL with ddH<sub>2</sub>O.</p>
<p><em><strong>5 M Ammonium acetate</strong></em><strong>:</strong><br />Dissolve 38.6 g ammonium acetate in 75 mL ddH<sub>2</sub>O, bring to 100 mL with ddH<sub>2</sub>O.</p>
<p><em><strong>180 mM Aluminium etc.</strong></em><strong>:</strong><br />Dissolve 8.2 g aluminium ammonium sulphate dodecahydrate in 75 mL ddH<sub>2</sub>O, bring to 100 mL with ddH<sub>2</sub>O.</p>
<p><em><strong>3% Calcium chloride</strong></em><strong>:</strong><br />Dissolve 3 g calcium chloride dihydrate in 75 mL ddH<sub>2</sub>O, bring to 100 mL with ddH<sub>2</sub>O.</p>
<p><em><strong>5.5 M Guanidine HCl</strong></em><strong>:</strong><br />Dissolve 52.6 g guanidine hydrochloride in 75 mL ddH<sub>2</sub>O, bring to 100 mL with ddH<sub>2</sub>O.</p>
<p><strong>Working solutions</strong></p>
<p>All working solutions are composites of stock solutions. All working solution compositions are given for a 100 mL final volume. However, working solutions may not be required in this amount. Some working solutions are designed to be easily created from combinations of stock solution volumes. For these solutions the number of volumes required per stock solution is given in brackets after the amount for 100 mL. Also note that some working solutions consist of a single stock solution.</p>
<p><em><strong>Lysis Solution</strong></em><strong>:</strong><br />To 75 mL ddH<sub>2</sub>O add 6.7 mL <em><strong>1 M Tris HCl (pH 8)</strong></em>, 5.3 mL <em><strong>0.5 M EDTA (pH 8)</strong></em>, 1.7 g guanidine thiocyanate, 8.7 g trisodium phosphate dodecahydrate and 0.2 g sodium chloride. Stir mixture until all solids dissolve. Adjust to pH 9.0 with 5 M HCl. Bring to final 100 mL volume with ddH<sub>2</sub>O.</p>
<p><strong><em>Soil Lysis Additive</em>:</strong><br />To 50 mL (8 volumes) <em><strong>180 mM Aluminium etc.</strong></em> add 43.75 mL (7 volumes) ddH<sub>2</sub>O and 6.25 mL (1 volume) <em><strong>20% SDS</strong></em>. Vortex briefly to mix.</p>
<p><strong><em>Tissue Lysis Additive</em>:</strong><br /><em><strong>20% SDS</strong></em></p>
<p><strong><em>Water Lysis Additive</em>:</strong><br />To 93.75 mL (15 volumes) ddH<sub>2</sub>O add 6.25 mL (1 volume) <em><strong>20% SDS</strong></em>. Vortex briefly to mix.</p>
<p><strong><em>Flocculant Solution</em>:</strong><br />To 50 mL (2 volumes) <em><strong>5 M Ammonium acetate</strong></em> add 25 mL (1 volume) <em><strong>180 mM Aluminium etc.</strong></em>. Vortex briefly before adding 25 mL (1 volume) <em><strong>3% Calcium chloride</strong></em>. Vortex briefly to mix.</p>
<p><strong><em>Binding Solution</em>:</strong><br /><em><strong>5.5 M Guanidine HCl</strong></em></p>
<p><strong><em>Tissue Binding Solution</em>:</strong><br />To 50 mL (1 volume) <em><strong>5.5 M Guanidine HCl</strong></em> add 50 mL (1 volume) 100% ethanol. Vortex briefly to mix.</p>
<p><strong><em>Wash Solution</em>:</strong><br />To 20 mL (2 volumes) ddH<sub>2</sub>O add 80 mL (8 volumes) 100% ethanol.</p>
<p><strong><em>Elution Buffer</em>:</strong><br />To 75 mL ddH<sub>2</sub>O add 1 mL <em><strong>1 M Tris HCl (pH 8)</strong></em> and 0.2 mL <em><strong>0.5 M EDTA (pH 8)</strong></em>. Bring to 100 mL with ddH<sub>2</sub>O.</p>

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