TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes (1) how to amplify the genomic markers SCAR3, SCAR4, SCAR5 and SCAR7 (developed by Thornhill et al., 2013) from genomic DNA of Aiptasia<em>,</em> (2) how to use Gibson Assembly® to clone them into a (pCR<sup>™</sup>II-TOPO<sup>®</sup>-derived) plasmid and (3) performing colony PCRs to screen for clones to sequence by Sanger sequencing. </p>
<p>The subcloning is highly recommended since strains might be heterozygous for some or all of the markers. </p>
<p> </p>
<p>SCAR: Aiptasia-specific inter-simple sequence repeat (ISSR)-derived sequence characterized amplified region markers.</p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p>Legal disclaimer:</p>
<p>GIBSON ASSEMBLY<sup>®</sup> is a registered trademark of Synthetic Genomics, Inc.</p>
<p>pCR<sup>™</sup> and TOPO<sup>®</sup> are trademarks of Thermo Fisher Scientific and its subsidiaries.</p>
<p>GoTaq<sup>®</sup> is a registered trademark of Promega Corporation.</p>
<p> </p>

[BEFORE_START]
<p>This protocol uses Gibson Assembly<sup>®</sup> for cloning the amplified SCAR markers into a PCR-amplified pCR<em>™</em>II-TOPO<sup>®</sup> back bone.</p>
<p>You need the following primers:</p>
<p> </p>
<p><strong>Table1</strong></p>
<table style="height: 246px; width: 2080px;">
<tbody>
<tr>
<td style="width: 177px;"> Forward primer</td>
<td style="width: 567px;">Sequence</td>
<td style="width: 27px;">Reverse primer</td>
<td style="width: 632px;">Sequence</td>
<td style="width: 1191px;">Function</td>
</tr>
<tr>
<td style="width: 177px;">ApSCAR3F</td>
<td style="width: 567px;">GAC CGT TAA AAT CTA AAG GTT TAC ATT AAG TAC AAG</td>
<td style="width: 27px;">ApSCAR3R</td>
<td style="width: 632px;">GGA TAA TGG TAG AGG GAA AGA AAG TGT CCG TAC</td>
<td style="width: 1191px;">Amplification of the <em>Aiptasia</em> SCAR3 marker.</td>
</tr>
<tr>
<td style="width: 177px;">ApSCAR4F</td>
<td style="width: 567px;">CTC ATC AGC CCA ATC AAA TTA GAC</td>
<td style="width: 27px;">ApSCAR4R</td>
<td style="width: 632px;">GTC CAA ATA TGA AAT AAT TAG ACT G</td>
<td style="width: 1191px;">Amplification of the <em>Aiptasia</em> SCAR4 marker.</td>
</tr>
<tr>
<td style="width: 177px;">ApSCAR5F</td>
<td style="width: 567px;">GCA AGT GTT TGT CTC AGC TTG TGT GAA TCT G</td>
<td style="width: 27px;">ApSCAR5R</td>
<td style="width: 632px;">CAC AAA GTT TCA TGC TCT TAG TAA GTT CAG</td>
<td style="width: 1191px;">Amplification of the <em>Aiptasia</em> SCAR5 marker.</td>
</tr>
<tr>
<td style="width: 177px;">ApSCAR7F</td>
<td style="width: 567px;">CCA TTC ATG ACG AAA TAG CTA TCC ACG TCC</td>
<td style="width: 27px;">ApSCAR7R2</td>
<td style="width: 632px;">GAT CTT AAA TAA TTA ATA ATA TAG CCA CAT ATT AAT CC</td>
<td style="width: 1191px;">Amplification of the <em>Aiptasia</em> SCAR7 marker.</td>
</tr>
<tr>
<td style="width: 177px;">SCAR3R_in_TOPOII_fw</td>
<td style="width: 567px;">GTA CGG ACA CTT TCT TTC CCT CTA CCA TTA TCC GGC GAA TTC TGC AGA TAT CC</td>
<td style="width: 27px;">SCAR3F_in_TOPOII_rev</td>
<td style="width: 632px;">CTT GTA CTT AAT GTA AAC CTT TAG ATT TTA ACG GTC AAG CTT GAT GCA TAG CTT GAG</td>
<td style="width: 1191px;">Amplification of a pCRII-TOPO back bone with flanks homologous to the SCAR3 amplicon allowing Gibson Assembly.</td>
</tr>
<tr>
<td style="width: 177px;">SCAR4R_in_TOPOII_fw</td>
<td style="width: 567px;">CAG TCT AAT TAT TTC ATA TTT GGA CGG CGA ATT CTG CAG ATA TCC</td>
<td style="width: 27px;">SCAR4F_in_TOPOII_rev</td>
<td style="width: 632px;">GTC TAA TTT GAT TGG GCT GAT GAG AAG CTT GAT GCA TAG CTT GAG</td>
<td style="width: 1191px;">Amplification of a pCRII-TOPO back bone with flanks homologous to the SCAR4 amplicon allowing Gibson Assembly.</td>
</tr>
<tr>
<td style="width: 177px;">SCAR5R_in_TOPOII_fw</td>
<td style="width: 567px;">CTG AAC TTA CTA AGA GCA TGA AAC TTT GTG GGC GAA TTC TGC AGA TAT CC</td>
<td style="width: 27px;">SCAR5F_in_TOPOII_rev</td>
<td style="width: 632px;">CAG ATT CAC ACA AGC TGA GAC AAA CAC TTG CAA GCT TGA TGC ATA GCT TGA G</td>
<td style="width: 1191px;">Amplification of a pCRII-TOPO back bone with flanks homologous to the SCAR5 amplicon allowing Gibson Assembly.</td>
</tr>
<tr>
<td style="width: 177px;">SCAR7R2_in_TOPOII_fw</td>
<td style="width: 567px;">GGA TTA ATA TGT GGC TAT ATT ATT AAT TAT TTA AGA TCG GCG AAT TCT GCA GAT ATC C</td>
<td style="width: 27px;">SCAR7F_in_TOPOII_rev</td>
<td style="width: 632px;">GGA CGT GGA TAG CTA TTT CGT CAT GAA TGG AAG CTT GAT GCA TAG CTT GAG</td>
<td style="width: 1191px;">Amplification of a pCRII-TOPO back bone with flanks homologous to the SCAR6 amplicon allowing Gibson Assembly.</td>
</tr>
</tbody>
</table>

[GUIDELINES]
<p>The PCR conditions are optimized for KOD (Hot Start) polymerase and work for SCAR3, SCAR4, SCAR5 and SCAR7 primers. </p>
<p><em>If you plan to subclone your SCAR marker fragments into plasmids before sequencing (and you should!), an effictive way to do this is Gibson Assembly<sup>®</sup>.</em></p>
<p><em>This method requires the fragments you want to assemble to have ≥20 bp homolog ends to each other. </em></p>
<p><em>To create linear plasmid fragments with flanks homolog to the respective SCAR primers do PCRs with the SCARx_in_TOPOII primer pairs from Table 1 on pCR™II-TOPO<sup>®</sup> plasmid DNA (or any plasmid derived thereof which has matching annealing sites for the primers).</em></p>
<p><em>I decided to keep the SCAR primers as they are and to add the homolog flanks to the plasmid backbone because the SCAR primers were already pretty sensitive to PCR conditions and PCRs on genomic DNA usually need more tweaking than on plasmid DNA.</em></p>
<p> </p>
<p>You can stop at any step of this protocol and store the samples at -20°C to continue later.</p>
<p> </p>
<p><strong>20x Borax running buffer for agarose gels:</strong></p>
<ul>
<li>Dissolve 38.18 g Sodium Tetraborate Decahydrate (Borax) in 900 ml distilled water (you might need to heat up the solution to dissolve everything)</li>
<li>Bring to 1 L with distilled water</li>
<li>DO NOT try adjusting the pH, it takes a looooot of boric acid, even with directly adding pure powder and the gels are running well without doing it.</li>
</ul>
<p>This results in a 100 mM solution, adjust the amount accordingly, when using other forms of Tetraborate (anhydrous etc.).</p>
<p> </p>
<p>Add 50 ml of the 20× buffer to 950 ml distilled water to obtain a 1× solution.</p>
<p> </p>
<div><a href="https://www.future-science.com/doi/abs/10.2144/04362BM02" target="_blank" rel="noopener noreferrer">Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis</a></div>
<div>Jonathan R. Brody and Scott E. Kern</div>
<p>BioTechniques 2004 36:2, 214-216</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.