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[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><strong>Prepare beads stock solution</strong></p>
<p> </p>
<p>This is the same beads stock solution as in the size selection protocol: https://www.protocols.io/view/dna-size-selection-1kb-and-clean-up-using-an-optim-ir3cd8n </p>
<p> </p>
<p> </p>
<p>For 10 mL:</p>
<p> </p>
<table style="width: 234px;">
<tbody>
<tr>
<td style="width: 102px;"> Final </td>
<td style="width: 57px;"> stock </td>
<td style="width: 58px;"> Input</td>
</tr>
<tr>
<td style="width: 102px;">10 mM Tris-HCl </td>
<td style="width: 57px;"> 1 M</td>
<td style="width: 58px;"> 100 μl</td>
</tr>
<tr>
<td style="width: 102px;">1 mM EDTA pH 8 </td>
<td style="width: 57px;">0.5 M </td>
<td style="width: 58px;"> 20 μl</td>
</tr>
<tr>
<td style="width: 102px;">1.6 M NaCl</td>
<td style="width: 57px;">5 M </td>
<td style="width: 58px;"> 3.2 ml</td>
</tr>
<tr>
<td style="width: 102px;">11% PEG 8000 </td>
<td style="width: 57px;">50% (w/v) </td>
<td style="width: 58px;"> 2.2 ml</td>
</tr>
<tr>
<td style="width: 102px;">0.4% beads (v/v)</td>
<td style="width: 57px;">100% </td>
<td style="width: 58px;"> 40 μl</td>
</tr>
<tr>
<td style="width: 102px;">Milliq Water </td>
<td style="width: 57px;"> </td>
<td style="width: 58px;"> 4.44 ml </td>
</tr>
</tbody>
</table>
<p><br /><em>*Because there were some questions considering the beads concentration:</em></p>
<p><em> </em></p>
<p><em>- 0.4% beads final are 40 µL of the Sera Mag speed beads in a total Volume of 10 mL 'beads stock solution'- thats not much but you definitely should see them in the solution! </em></p>
<p><em> </em></p>
<p><em>- there is no issue in increasing the beads concentration to e.g. 1% (100 µL in the final 10 mL), there should be no change in size selection, the beads have a  binding capacity of  5 µg/ml anyway. I tested the different beads concentrations and there was no difference between final 0.4% - 2% beads, I just took the 0.4% because I needed a lot of beads solution in total. </em></p>
<p> </p>
<p><em>-Make sure to use the Sera-Mag SpeedBeads and not the AMPure XP beads for preparing the  beads stock solution. The AMPure XP beads are used straight (at 0.45V)  without further manipulation. </em></p>
<p> </p>
<p> </p>
<p> </p>
<p>1. Frist combine only Water, Tris-HCl, EDTA and NaCl in a 50 mL tube. </p>
<p> </p>
<p>2. Vortex Sera-Mag SpeedBeads (GE Healthcare, 65152105050250) very well and pipette 40 μl into a 1.5 ml tube, put it on the magnetic rack and wait until solution has cleared up and all beads have bound to the back of the tube </p>
<p> </p>
<p>3. Wash beads by removing supernatant and adding 1.5 ml milliq water</p>
<p> </p>
<p>4. Take tube of the magnet, mix well, spin down in a microcentrifuge and put back on the magnet </p>
<p> </p>
<p>5. Wait for beads to assemble at the back of the tube </p>
<p> </p>
<p>6. Pipette off and discard supernatant</p>
<p> </p>
<p>7. Repeat washing (steps 3 - 6) 3 more times </p>
<p> </p>
<p>8. After pipetting of the supernatant the last time take off tube from the magnet and add 40 μl of the previous (step 1) prepared stock solution, mix well, spin down and pipette everyting into the remaining stock solution in the 50 mL tube and mix</p>
<p> </p>
<p>9.  Now the 2.2 ml 50% PEG can be added to the stock solution, which after vortexing very well is ready for use. </p>
<p> Be careful to actually pipette 2.2 ml as solution is very viscous, but the final concentration of PEG is crucial for the clean up to work properly.</p>
<p> </p>
<p> </p>
<p> </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 <br />(stock solutions)</strong></p>
<p> </p>
<p><strong>Lysis buffer:</strong></p>
<p>10% PVP 40 </p>
<p>10% PVP 10</p>
<p>5M NaCl </p>
<p>1M TRIS pH 8 </p>
<p>0.5 M EDTA</p>
<p>20% SDS</p>
<p>Sodium metabisulfite (190.1 g/mol)</p>
<p>Molecular biology grade water</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))  very useful tips about preparing the high concentrated PEG solutions can be found - definitely worth a look! </p>
<p> </p>
<p>Sera-Mag SpeedBeads  (GE Healthcare, 65152105050250) </p>
<p>50% PEG 8000</p>
<p>5 M NaCl</p>
<p>1 M Tris-HCl pH 8</p>
<p>0.5 M EDTA pH 8 </p>
<p>molecular biology grade water </p>
<p> </p>
<p> </p>
<p><strong>Other solutions</strong>:</p>
<p>5M Potassium Acetate  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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