TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The isolation of nuclear genomic DNA of high molecular weight is becoming a crucial step for obtaining long read sequencing data produced by the PacBio and Oxford Nanopore platforms. Although it involves a few more steps than a standard total genomic DNA preparations, it is worth optimizing the protocol to avoid wasting valuable sequence data on organellar DNA. The current protocol has been successfully applied to kiwifruit, apple, mānuka, <em>Nicotiana</em> <em>benthamiana</em>, some solanaceas, and two insects (pupae of the light brown apple moth (<em>Epiphyas postvittana</em>) and hind leg of a weta (<em>Deinacrida </em>spp)). Three alternative methods to extract DNA are also presented.</p>

[BEFORE_START]
<p><strong>Solutions and Reagents</strong></p>
<ul>
<li>NEB (Nuclei Extraction Buffer) 0.5 M Mannitol, 10 mM PIPES-KOH, 10 mM MgCl<sub>2</sub> 6H<sub>2</sub>O, 2% PVP K40, 200 mM L-lysine monohydrochloride, 6 mM EGTA, pH 6</li>
</ul>
<table style="width: 433px;">
<tbody>
<tr>
<td style="width: 206px;">
<p style="text-align: left;">Reagent</p>
</td>
<td style="text-align: left; width: 150px;">
<p>Sigma Aldrich cat #</p>
</td>
<td style="text-align: center; width: 59px;">
<p>1 L</p>
</td>
</tr>
<tr style="text-align: left;">
<td style="width: 206px;">
<p>Mannitol</p>
</td>
<td style="width: 150px;">
<p>M4125-1KG</p>
</td>
<td style="text-align: right; width: 59px;">
<p>91 g</p>
</td>
</tr>
<tr style="text-align: left;">
<td style="width: 206px;">
<p>PIPES-KOH</p>
</td>
<td style="width: 150px;">
<p>P7643-100G</p>
</td>
<td style="text-align: right; width: 59px;">
<p>3.78 g</p>
</td>
</tr>
<tr style="text-align: left;">
<td style="width: 206px;">
<p>MgCl<sub>2</sub> 6H<sub>2</sub>O</p>
</td>
<td style="width: 150px;">
<p>M9272-100G</p>
</td>
<td style="text-align: right; width: 59px;">
<p>2.03 g</p>
</td>
</tr>
<tr style="text-align: left;">
<td style="width: 206px;">
<p>PVP K40</p>
</td>
<td style="width: 150px;">
<p>PVP40-500G</p>
</td>
<td style="text-align: right; width: 59px;">
<p>20 g</p>
</td>
</tr>
<tr style="text-align: left;">
<td style="width: 206px;">
<p>L-lysine monohydrochloride</p>
</td>
<td style="width: 150px;">
<p>L5626-1KG</p>
</td>
<td style="text-align: right; width: 59px;">
<p>36.52 g</p>
</td>
</tr>
<tr style="text-align: left;">
<td style="width: 206px;">
<p>EGTA</p>
</td>
<td style="width: 150px;">
<p>E3889-100G</p>
</td>
<td style="text-align: right; width: 59px;">
<p>2.28 g</p>
</td>
</tr>
</tbody>
</table>
<p style="text-align: left;"> </p>
<p style="text-align: left;">Heat up 800 mL of deionized water, 2-4 min in microwave. Pour into a glass beaker with a magnetic stirrer. Add PVP K40, three spoonfuls at a time, stir vigorously. To speed up this process you can use two flat spatulas and “cut” the PVP blobs as if it was a steak.</p>
<p style="text-align: left;">Add mannitol and dissolve completely before adding PIPES, magnesium, lysine, and EGTA. Adjust the pH to 6.0 with 10 M NaOH. Adjust the volume to 1 L. Split the buffer into two 1 L Schott bottles. Autoclave and cool down to room temperature. Store at 4°C until ready to use.</p>
<ul style="text-align: left;">
<li>NEB complete and NEB –no b-mercaptoethanol</li>
</ul>
<p style="text-align: left;">Just before starting the nuclei isolation add 0.9 g sodium meta-bisulphite (Sigma Aldrich cat # 255556-100G) to <u>each</u> Schott bottle. Close the bottle and shake until it is completely dissolved. The final concentration is 10 mM.</p>
<p style="text-align: left;">Add 0.2 mL β-mercaptoethanol to <u>one Schott bottle only</u>. Label this one as NEB complete buffer. The other flask is NEB – no β-mercaptoethanol. Keep on ice.</p>
<ul style="text-align: left;">
<li>25% Triton X-100 in NEB complete</li>
</ul>
<p style="text-align: left;">Transfer 7.5 mL of NEB complete buffer to a 50 mL Falcon tube. Add 2.5 mL of Triton X-100, use a wide bore tip. Aspirate the detergent very slowly from the stock bottle until you don’t see any change in the liquid level. Do not dip the tip too far into the stock bottle to avoid carrying extra detergent into the Falcon tube. Dispense the detergent into the 7.5 mL of buffer, do it very slowly. Aspirate some buffer to clean up the inside of the wide bore tip until there is no more detergent lumps visible. Close the tube and vortex thoroughly until fully dissolved. Spin down ~ 10 min at 3000 rpm at room temperature to pop all the bubbles. Keep the tube on ice.</p>
<ul style="text-align: left;">
<li>Tris-borate/K-acetate/SDS Lysis buffer:<strong> </strong>0.5% SDS, 5 mM EDTA, 150 mM Tris-borate pH 7.4</li>
</ul>
<p style="text-align: left;">To prepare this solution the Tris buffer is titrated with boric acid, instead of the usual HCl. Autoclave and keep at room temperature.</p>
<ul>
<li style="text-align: left;">CTAB/NaCl lysis buffer: 2% CTAB (hexadecyltrimethyl ammonium bromide), 2% PVP K40 (polyvinyl pyrrolidinone K40), 2 M NaCl, 25 mM EDTA, 100 mM Tris-HCl pH 8.0. To dissovle the PVP lumps, use the same technique as described above for NEB. Split the buffer into two 1 L Schott bottles and autoclave. Keep at room temperature.</li>
<li>5 M potassium acetate pH 7.0. Prepare according to <a href="http://www.molecularcloning.com/" target="_blank" rel="noopener noreferrer">Green and Sambrook 2012</a> and sterilize by filtration.</li>
<li>100% Ethanol</li>
<li>70% Ethanol</li>
<li>Isopropanol</li>
<li>Chloroform:isoamyl alcohol (24:1)</li>
<li>3 M sodium acetate pH 5.2 (see <a href="http://www.molecularcloning.com/" target="_blank" rel="noopener noreferrer">Green and Sambrook 2012</a>)</li>
<li>β-mercaptoethanol (Sigma Aldrich cat # 63689)</li>
<li>TE pH 7.5 (10 mM Tris-HCl, 1 mM EDTA pH 7.5)</li>
<li>RNase A 100 mg/mL (Qiagen cat # 19101)</li>
<li>Proteinase K 20 mg/mL (Qiagen cat # 19133)</li>
<li>Qiagen DNeasy Plant Maxi kit (Qiagen cat # 68163)</li>
<li>SybrSafe (Invitrogen cat # S33102)</li>
<li>Lambda DNA (Invitrogen cat # SD0011)</li>
<li>1 kb+ ladder (Invitrogen cat # 10787018)</li>
<li>Lambda ladder (BioRad cat # 170-3635)</li>
<li>Standard agarose (Invitrogen cat # 16500500)</li>
<li>PFGE grade agarose (BioRad cat # 1613108)</li>
<li>1X TAE gel running buffer</li>
</ul>
<p style="text-align: left;"> </p>
<p style="text-align: left;"> <strong>Equipment</strong></p>
<p style="text-align: left;"> </p>
<ul style="text-align: left;">
<li>Waring commercial blender or a standard kitchen blender (for example, Sunbeam model PB7950)</li>
<li>Miracloth</li>
<li>Cheese cloth, sterile</li>
<li>Two Large funnels</li>
<li>Two 500 mL cylinder</li>
<li>Parafilm</li>
<li>Wide bore tips: 200, 1000 and 5000 μL, sterile, to pipette the purified nuclear genomic DNA and the Triton X-100 stock solution</li>
<li>Kimwipes</li>
<li>Qubit system with HS dsDNA kit or DeNovix (DS-11 FX, FX+ or QFX) with dsDNA High Sensitivity kit</li>
<li>Microvolume spectrophotometer Nanodrop or DeNovix (DS-11, 11+, FX or FX+)</li>
<li>Sorvall centrifuge or similar with SLA 600 TC rotor or SS34 rotor</li>
<li>Nalgene centrifuge tubes Nalgene™ Oak Ridge (cat # 3119-0050)</li>
<li>Bench top centrifuge with plate rotor (for example, Eppendorf SKU 5810000084 Rotor A-4-62, with 4 MTP buckets; Cat. # 5810711002)</li>
<li>Falcon tubes, sterile, 50 mL</li>
<li>Microcentrifuge</li>
<li>1.5 mL microcentrifuge tubes, sterile</li>
<li>Gel electrophoresis box and power supply</li>
<li>CHEF-DR III variable angle system for PFGE analysis (BioRad cat # 1703702)</li>
<li>Water bath with shaker</li>
</ul>
<p><strong>Acknowledgements</strong></p>
<p> </p>
<p>The method has been improved thanks to my colleagues' interest in learning it: Paul Datson, Don Hunter, Mark Livermore, Elena Lopez Girona, John McCallum, Fatima Naim, Craig Woods, Melinda Zhang, and Qiong Zhang.</p>

[GUIDELINES]
<p>Each plant poses a different challenge regarding the best approach to extract good quality DNA. The following review gives a very thorough introduction on this topic: Kasem S, Rice N, Henry R (2008) DNA extraction from plant tissue. Plant Genotyping II: SNP Technology. pp. 219-271.</p>
<p>The method presented here has been applied in a mid-throughput manner using a blender to homogenize the plant tissue instead of the traditional freeze/grind method with liquid nitrogen. However if the leaf tissue is tough (for example mānuka or rosemary) it would be best to use liquid nitrogen and a precooled mortar and pestle. The same would apply to very precious samples where nothing should be wasted.</p>

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