TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Third generation sequencing aims to sequence long DNA molecules. High-quality and high-molecular-weight DNA is needed to fully benefit from the potential of third-generation sequencers. The cost of sequencing continues to decrease but DNA extraction kits remain expensive. Here is a fast and cheap protocol to extract genomic DNA from Gram-negative bacteria. The original protocol has been fine-tuned for Xanthomonas ssp., which is a tricky bacteria as it secrets a lot of extracellular polysaccharides. We found that bacterial culture can impact the purity of the final DNA solution. In addition, the lysis temperature and mixing by pipetting are critical to get an efficient homogenization as the sample becomes very viscous at some steps (see 'Assessment of various parameters.xlsx' attached below).</p>
<p>High yield Nanopore runs can be achieved with these gDNA samples (see 'Nanopore sequencing report.pdf' attached below).</p>
<p> </p>
<p>Acknowledgements:</p>
<p>I would like to thank Harihar Jaishree Subrahmaniam, Rekha Gopalan Nair, and BenjaminSchwessinger for their feedbacks and their corrections on this protocol.</p>
<p> </p>

[BEFORE_START]
<p><strong>Prepare the following solutions:</strong></p>
<ul>
<li><strong>Lysis buffer:</strong></li>
</ul>
<table width="1060">
<tbody>
<tr>
<td width="403">Lysis buffer</td>
<td width="366">Stock concentration</td>
<td width="135">Final concentration</td>
<td width="156">Volume for 10ml </td>
</tr>
<tr>
<td width="403">H2O</td>
<td width="366">-</td>
<td width="135">-</td>
<td width="156">5.25 ml</td>
</tr>
<tr>
<td width="403">NaCl</td>
<td width="366">2.5 M</td>
<td width="135">0.5M</td>
<td width="156">2ml</td>
</tr>
<tr>
<td width="403">Tris HCl pH=8</td>
<td width="366">1 M</td>
<td width="135">100mM</td>
<td width="156">1ml</td>
</tr>
<tr>
<td width="403">EDTA pH=8 0</td>
<td width="366">0.5 M</td>
<td width="135">50mM</td>
<td width="156">1ml</td>
</tr>
<tr>
<td width="403">SDS</td>
<td width="366">20%</td>
<td width="135">1.5%</td>
<td width="156">
<p>750µl</p>
</td>
</tr>
<tr>
<td width="403">Total</td>
<td width="366">-</td>
<td width="135">-</td>
<td width="156">
<p>10ml</p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<ul>
<li><strong>Beads solution:</strong></li>
</ul>
<p>Beads solution is the same as in this protocol:</p>
<p> </p>
<div>
<div>Ramawatar, Benjamin Schwessinger (1523238626). DNA size selection (&gt;3-4kb) and purification of DNA using an improved homemade SPRI beads solution.. <strong>protocols.io</strong>
<div>dx.doi.org/10.17504/protocols.io.n7hdhj6</div>
<div>
<div> </div>
<div>
<table width="1060">
<tbody>
<tr>
<td width="403">Beads solution</td>
<td width="366">Stock concentration</td>
<td width="135">Final concentration</td>
<td width="156">Volume for 10ml </td>
</tr>
<tr>
<td width="403">Tris HCl pH=8</td>
<td width="366">1 M</td>
<td width="135">10 mM</td>
<td width="156"> 100 μl</td>
</tr>
<tr>
<td width="403">EDTA pH=8 0</td>
<td width="366">0.5 M </td>
<td width="135">1 mM</td>
<td width="156"> 20 μl</td>
</tr>
<tr>
<td width="403">NaCl</td>
<td width="366">5 M </td>
<td width="135">1.6 M</td>
<td width="156">3.2 ml</td>
</tr>
<tr>
<td width="403">PEG 8000 </td>
<td width="366">50% (w/v) </td>
<td width="135">11%</td>
<td width="156">2.2 ml</td>
</tr>
<tr>
<td width="403"> Tween 20</td>
<td width="366"> 10 % (v/v)</td>
<td width="135">0.20%</td>
<td width="156">200 µl</td>
</tr>
<tr>
<td width="403">Washed Sera-Mag beads (v/v)</td>
<td width="366">100% </td>
<td width="135">0.40%</td>
<td width="156"> 40 μl</td>
</tr>
<tr>
<td width="403">H2O</td>
<td width="366"> -</td>
<td width="135">-</td>
<td width="156">up to 10ml</td>
</tr>
<tr>
<td width="403">Total </td>
<td width="366"> -</td>
<td width="135">-</td>
<td width="156">
<p> 10 ml</p>
</td>
</tr>
</tbody>
</table>
</div>
</div>
<div> </div>
<div>1- Frist combine only Water, Tris-HCl, EDTA and NaCl in a 50 mL tube. </div>
<div> </div>
<div>2- Vortex Sera-Mag SpeedBeads® Carboxyl Magnetic Beads (GE Healthcare)  very well and pipette 40 μl into a 1.5 ml tube, put it on the magnetic rack and wait until the solution has cleared up and all beads have bound to the back of the tube</div>
<div> </div>
<div>3- Wash beads by removing supernatant and adding 1 ml Milli-Q water</div>
<div> </div>
<div>4- Take tube of the magnet, mix well, spin down in a microcentrifuge and put back on the magnet</div>
<div> </div>
<div>5- Wait for beads to assemble at the back of the tube </div>
<div> </div>
<div>6- Pipette off and discard supernatant</div>
<div> </div>
<div>7- Repeat washing (steps 3 - 6) 3 more times</div>
<div>
<div>
<div>
<div id="">
<div>
<div>
<div>
<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 everything 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>
<div>
<div>
<div id="">
<div>
<div>
<div>
<p> </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>10-</p>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
<ul>
<li><strong>Potassium acetate 5M:</strong></li>
</ul>
<div>Dissolve 4.9 g of potassium acetate in 10 ml of molecular grade water.</div>
<div> </div>
<div> </div>
<div> </div>
<div> </div>
<div> </div>
<div> </div>
<div> </div>
<div> </div>
<div> </div>
<div> </div>
<div> </div>
<div> </div>
<div> </div>
<div> </div>
<div> </div>
<div> </div>
<div> </div>
</div>
</div>

[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><a href="https://www.future-science.com/doi/10.2144/000114460" target="_blank" rel="noopener noreferrer">https://www.future-science.com/doi/10.2144/000114460</a></p>
<p> </p>
<p>The beads solution used in this protocol is the same as in this protocol:</p>
<div>
<div>Ramawatar, Benjamin Schwessinger (1523238626). DNA size selection (&gt;3-4kb) and purification of DNA using an improved homemade SPRI beads solution.. <strong>protocols.io</strong>
<div><a href="dx.doi.org/10.17504/protocols.io.n7hdhj6" target="_blank" rel="noopener noreferrer">dx.doi.org/10.17504/protocols.io.n7hdhj6</a></div>
</div>
</div>
<p> </p>
<p>When citing please make sure to mention the original Mayjonade et al. protocol as described above and the protocol for the beads solution.</p>
<p> </p>
<p><span style="font-weight: 400;">pH makes a huge difference in the solubility of DNA and beads. If not set properly, beads tend to clump at DNA elution step that could lose as much 60-70 percent of the DNA. So make sure that your elution buffer (e.g. 10 mM Tris or 0.1 x TE) has a pH of 8. </span></p>
<p> </p>
<p><span style="font-weight: 400;">Store beads solution at 4°C. Allow beads solution to come to room temperature before use (30 minutes).</span></p>
<p> </p>
<p><span style="font-weight: 400;">Mix the beads solution well by vortexing before use.</span></p>
<p> </p>
<p><span style="font-weight: 400;">Always use freshly prepared  70 % Ethanol </span></p>
<p> </p>

[STEPS]
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