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[DESCRIPTION]
<p>Optical mapping technologies assist the assembly of large and complex genomes. Although protocols for preparing this kind of samples are now conveniently available in kits, some plants won't respond to the standardized conditions or the researcher might not have access to it.<br />
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<div class="nova-e-text nova-e-text--size-m nova-e-text--family-sans-serif nova-e-text--spacing-none message-body-part">Here I provide a draft protocol I have been working on for kiwifruit, with limited success. The nuclear genomic DNA produced is ~ 1 Mbp long but it has not passed the service provider QC step.</div>
<div class="nova-e-text nova-e-text--size-m nova-e-text--family-sans-serif nova-e-text--spacing-none message-body-part">I would be very happy to hear from the ResearchGate and Protocos.io communities regarding any interest in collaborating to improve this method and eventually make it available to a wider audience.</div>
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<p> </p>

[BEFORE_START]
<h3>Prepare the following buffers:</h3>
<h3>1.      Nuclei isolation </h3>
<ul>
<li>Nuclei isolation buffer (Otto I)</li>
</ul>
<p>0.1 M citric acid, 0.5% Tween 20 (v/v), pH 2-3, prepared in sterile deionized water, cooled on ice.</p>
<p> </p>
<ul>
<li>Nuclei extraction buffer (NEB) Complete</li>
</ul>
<p>0.5 M Mannitol, 10 mM PIPES-KOH pH 6, 10 mM MgCl<sub>2</sub>, 2% PVP-K40, 200 mM L-lysine monohydrochloride, 6 mM EGTA, 10 mM sodium metabisulfite and 5 mM β-mercaptoethanol (in percentage v/v is equal to 0.04%).</p>
<p> </p>
<table>
<tbody>
<tr>
<td width="208">
<p> </p>
</td>
<td width="142">
<p>g/L</p>
</td>
</tr>
<tr>
<td width="208">
<p>Mannitol</p>
</td>
<td width="142">
<p>91</p>
</td>
</tr>
<tr>
<td width="208">
<p>PIPES-KOH</p>
</td>
<td width="142">
<p>3.78</p>
</td>
</tr>
<tr>
<td width="208">
<p>MgCl<sub>2</sub>.7H<sub>2</sub>0</p>
</td>
<td width="142">
<p>2</p>
</td>
</tr>
<tr>
<td width="208">
<p>L-lysine monohydrochloride</p>
</td>
<td width="142">
<p>36.5</p>
</td>
</tr>
<tr>
<td width="208">
<p>EGTA</p>
</td>
<td width="142">
<p>2.28</p>
</td>
</tr>
<tr>
<td width="208">
<p>PVP-K40</p>
</td>
<td width="142">
<p>20</p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p>Adjust to pH 6 with 10 M NaOH, split in two 1 L bottles, and autoclave, store at 4°C</p>
<p> </p>
<p>Before using this buffer, add 0.1 g of sodium metabisulfite to two 50 mL aliquots, in Falcon tubes. Add 20 µL β-mercaptoethanol to only one tube, now labelled as NEB Complete. </p>
<ul>
<li>25% Triton X-100</li>
</ul>
<p>750 µL NEB complete plus 250 µL mL Triton X-100. Vortex and spin down until all is dissolved. </p>
<ul>
<li>NEB - βME</li>
</ul>
<p>The other 50 mL NEB tube containing sodium metabisulfite, but no β-mercaptoethanol.</p>
<p> </p>
<h3>2.      Percoll gradient buffers </h3>
<ul>
<li>NEB -PVP/M/T</li>
</ul>
<p>0.5 M Mannitol, 10 mM PIPES-KOH, 10 mM MgCl<sub>2</sub>, 200 mM L-lysine monohydrochloride, 6 mM EGTA, pH 7.0, sterile </p>
<ul>
<li>Percoll GE Healthcare, cat# 17-0891-01. Once opened, store at 4°C. </li>
</ul>
<ul>
<li>NEB-A</li>
</ul>
<table>
<tbody>
<tr>
<td width="142">
<p>NEB -PVP/M/T</p>
</td>
<td width="80">
<p>50 mL</p>
</td>
</tr>
<tr>
<td width="142">
<p>Na-metabisulfite</p>
</td>
<td width="80">
<p>0.1 g</p>
</td>
</tr>
<tr>
<td width="142">
<p>β-mercaptoethanol</p>
</td>
<td width="80">
<p>100 µL</p>
</td>
</tr>
<tr>
<td width="142">
<p>Triton X-100</p>
</td>
<td width="80">
<p>250 µL</p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<ul>
<li>NEB-B</li>
</ul>
<table>
<tbody>
<tr>
<td width="132">
<p>NEB-PVP/M/T</p>
</td>
<td width="76">
<p>50 mL</p>
</td>
</tr>
<tr>
<td width="132">
<p>Na-metabisulfite</p>
</td>
<td width="76">
<p>0.1 g</p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<h3>3.      Agarose plugs </h3>
<ul>
<li>CleanCut 2% Agarose, BioRad cat# 1703594</li>
<li>50-Well Disposable Plug Molds, BioRad #1703713 </li>
</ul>
<h3>4.      Proteinase K digestion solutions </h3>
<ul>
<li>2X Proteinase K lysis buffer (2X TLS), sterile</li>
</ul>
<p>20 mM Tris-HCl pH 8, 400 mM L-lysine monohydrochloride, 2% lauryl sarcosine, pH 8.0</p>
<ul>
<li>Proteinase K (20 mg/mL, Qiagen cat# 19133) </li>
</ul>
<ul>
<li>Proteinase K lysis mix</li>
</ul>
<table>
<tbody>
<tr>
<td width="132">
<p>2X TLS</p>
</td>
<td width="77">
<p>3 mL</p>
</td>
</tr>
<tr>
<td width="132">
<p>0.5 M EDTA pH 8</p>
</td>
<td width="77">
<p>3 mL</p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p>Prepare fresh by adding 396 µL Proteinase K</p>
<p> </p>
<ul>
<li>Wash-1</li>
</ul>
<table>
<tbody>
<tr>
<td width="133">
<p>2X TLS</p>
</td>
<td width="77">
<p>25 mL</p>
</td>
</tr>
<tr>
<td width="133">
<p>0.5 M EDTA pH 8</p>
</td>
<td width="77">
<p>2.5 mL</p>
</td>
</tr>
<tr>
<td width="133">
<p>ddH<sub>2</sub>O</p>
</td>
<td width="77">
<p>22.5 mL</p>
</td>
</tr>
<tr>
<td width="133">
<p>Na-metabisulfite</p>
</td>
<td width="77">
<p>0.1 g</p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<ul>
<li>Wash-2</li>
</ul>
<table>
<tbody>
<tr>
<td width="124">
<p>2X TLS</p>
</td>
<td width="87">
<p>25 mL</p>
</td>
</tr>
<tr>
<td width="124">
<p>0.5 M EDTA pH 8</p>
</td>
<td width="87">
<p>0.5 mL</p>
</td>
</tr>
<tr>
<td width="124">
<p>ddH<sub>2</sub>O</p>
</td>
<td width="87">
<p>24.5 mL</p>
</td>
</tr>
</tbody>
</table>
<p> </p>

[GUIDELINES]
<p><strong>Consumables and equipment</strong></p>
<ul>
<li>Sprouted kiwifruit canes grown in the glasshouse, with young leaves and plenty of roots</li>
<li>Scissors</li>
<li>Fine hair paint brush</li>
<li>Scalpel blades</li>
<li>Plastic Petri Dishes</li>
<li>Tweezers</li>
<li>15 mL Falcon tubes with rack</li>
<li>50 mL Falcon tubes with rack</li>
<li>Tissue homogenizer, ~ 7 mL capacity</li>
<li>Disposable plastic Pasteur pipettes</li>
<li>Glass Pasteur pipettes with bulb</li>
<li>Miracloth sheets cut 10 x 10 cm</li>
<li>Rubber bands</li>
<li>Spatulas with flat and round end points</li>
<li>SS34 rotor with sterile OakRidge tubes, and centrifuge, precooled at 10°C</li>
<li>Falcon cell strainers, 100 and 40 µm (Fisher Scientific cat# 08-771-19 and 08-771-1)</li>
<li>Benchtop centrifuge with swing bucket rotor, precooled at 10°C</li>
<li>Benchtop microcentrifuge with 1.5 mL Eppendorf and screw-cap tubes</li>
<li>Water bath at 70°C and hot block calibrated at 43°C</li>
<li>Hybridization oven set at 50°C</li>
</ul>

[STEPS]
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