TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Extraction of high quality DNA for long read sequencing e.g. the Oxford Nanopore </p>
<p>Optimized for DNA extraction from eucalyptus grandis and eucalyptus pauciflora. </p>
<p> </p>
<p>This protocol contains an optional Chloroform clean up step which is necessary for eucalyptus but might not be for other tissue. </p>
<p> </p>
<p>For long DNA fragments don't vortex the DNA sample. </p>
<p> </p>

[BEFORE_START]
<p>Prepare stock of beads solution:</p>
<p> </p>
<table>
<tbody>
<tr>
<td> Final </td>
<td> stock </td>
<td> Input</td>
</tr>
<tr>
<td>10 mM Tris-HCl </td>
<td> 1 M</td>
<td> 100 μl</td>
</tr>
<tr>
<td>1 mM EDTA pH 8 </td>
<td>0.5 M </td>
<td> 20 μl</td>
</tr>
<tr>
<td>1.6 M NaCl</td>
<td>5 M </td>
<td> 3.2 ml</td>
</tr>
<tr>
<td>11% PEG 8000 </td>
<td>50% (w/v) </td>
<td> 2.2 ml</td>
</tr>
<tr>
<td>0.4% beads (v/v)</td>
<td>100% </td>
<td> 40 μl</td>
</tr>
<tr>
<td>Milliq Water </td>
<td> </td>
<td> 4.44 ml </td>
</tr>
</tbody>
</table>
<p> </p>
<p>For exact preparation of beads solution please look at: https://www.protocols.io/view/dna-size-selection-1kb-and-clean-up-using-an-optim-ir3cd8n</p>
<p> </p>
<p>Always use fresh lysis buffer and fresh 70 % Ethanol </p>
<p> </p>
<p>Clean up steps 26 - 41 are optional and are only necessary in recalcitrant tissue like eucalyptus. The extraction and clean up step can also be done on seperate days if DNA is stored at 4°C. </p>

[GUIDELINES]
<p>Modified from the protocol of Baptiste Mayjonade, Jérome Gouzy, Cécile Donnadieu, Nicolas Pouilly, William Marande, Caroline Callot, Nicolas Langlade and Stéphane Munos, High molecular weight gDNA extraction, Bio Techniques, Vol. 61, No. 4, October 2016, pp. 203-205. </p>
<p> </p>
<p>Link to webpage: http://www.biotechniques.com/BiotechniquesJournal/2016/October/Extraction-of-high-molecular-weight-genomic-DNA-for-long-read-sequencing-of----single-molecules/biotechniques-365135.html </p>
<p> </p>
<p>When citing please make sure to also mention the original Mayjonade et al. protocol as described above. </p>
<p> </p>
<p>Thank you Benjamin Schwessinger for the constant support in the lab and outside! </p>
<p> </p>
<p><strong>Reagents required</strong></p>
<p> </p>
<p><strong>Lysis buffer:</strong></p>
<p>1% PVP 40 </p>
<p>1% PVP 10</p>
<p>500 mM NaCl </p>
<p>100 mM TRIS pH 8 </p>
<p>500 mM EDTA</p>
<p>1.25% SDS</p>
<p>1% Sodium metabisulfite </p>
<p>Adjust with molecular biology grade water to desired volume </p>
<p> </p>
<p><strong>Beads solution</strong> (adapted from Philippe Jolivet and Joseph W. Foley, 2015 - Solutions for purifying nucleic acides by solid-phase reversible immobilization (SPRI))</p>
<p> </p>
<p>0.4% Sera-Mag SpeedBead magnetic carboxylate modified particles (wash 4 times with water to remove sodium azide)</p>
<p>11% PEG 8000</p>
<p>1.6 M NaCl</p>
<p>10 mM Tris-HCl pH 8</p>
<p>1 mM EDTA pH 8 </p>
<p>adjuste with molecular biology grade water to desired volume</p>
<p> </p>
<p> </p>
<p><strong>Other solutions</strong>:</p>
<p>Potassium Acetate 5M pH 7.5 </p>
<p>Ethanol 70%</p>
<p>TE-Buffer (10 mM Tris, 1 mM EDTA)</p>
<p> </p>
<p>Additional for Clean up step: </p>
<p>Chloroform:Isoamylalcohol (24:1)</p>
<p>Natriumacetate 3 M </p>
<p>Ethanol 100 % </p>
<p>Ethanol 70 %</p>
<p>TE-Buffer </p>
<p> </p>
<p><strong>Enzymes</strong></p>
<p>RNAse A (1000 U/ml, Thermo Fisher EN0541)</p>
<p>Proteinase K (800U/ml, NEB P81072)</p>
<p> <br />Materials: </p>
<p>TissueLyser II (Qiagen)</p>
<p>Thermomixer</p>
<p>Magnetic rack for Eppendorf tubes</p>
<p> </p>

[STEPS]
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