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[DESCRIPTION]
<ol>
<li><strong> DNA Extraction</strong></li>
</ol>
<ul>
<li>Extract DNA from sample using the phenol/chloroform procedure or your kit of choice. We typically use the Mo Bio Power Soil DNA extraction kit for extracting DNA from soil and plant litter samples.</li>
<li>Use the Nanodrop in the Martiny lab to estimate the concentration and purity of your DNA extraction.</li>
<li>Dilute DNA to 10 ng/ul prior to PCR.</li>
</ul>
<p> </p>
<p> </p>
<ol start="2">
<li><strong> PCR Amplification</strong></li>
</ol>
<p><u>Pre-amplification checklist:</u></p>
<ul>
<li>All pipetting should be done with aerosol barrier, PCR certified pipet tips in a PCR work station</li>
<li>On ice thaw primers, DNA and PCR master mix</li>
<li>Decontaminate pipettors and interior surfaces of a PCR workstation with DNA/RNA Away or 10% bleach solution.</li>
<li>UV irradiate PCR work station interior, pipettors and consumables for minimum 15 min</li>
<li>All samples should be amplified in triplicate.</li>
<li>Include negative (no template) and positive (DNA isolated from mushroom) controls.</li>
</ul>
<p> </p>
<p><u>Primer information:</u></p>
<p>Forward primer:</p>
<p>454 primer B + 'AG' linker + SSU817f</p>
<p>GCCTTGCCAGCCCGCTCAGAGTTAGCATGGAATAATRRAATAGGA</p>
<p> </p>
<p>Reverse Primer:</p>
<p>454 primer A + 12-base bar code + 'AC' linker + ssu1196r</p>
<p>GCCTCCCTCGCGCCATCAG-12 base bar code-ACTCTGGACCTGGTGAGTTTCC</p>
<p>Example barcode: ACACACTATGGC</p>
<p>Example primer sequence: GCCTCCCTCGCGCCATCAGACACACTATGGCACTCTGGACCTGGTGAGTTTCC</p>
<p> </p>
<p><u>PCR Cocktail:</u></p>
<p>22.5 ul PCR master mix (we use the Invitrogen Platinum PCR SuperMix)</p>
<p>1 ul BSA</p>
<p>0.75 ul 10 uM forward primer (all samples get the same forward primer)</p>
<p>0.75 ul 10 uM reverse primer (all samples get a unique barcoded reverse primer)</p>
<p>1 ul template DNA</p>
<p> </p>
<p><u>PCR cycle:</u></p>
<p>Samples are initially denatured at 94°C for 10 min, then amplified using 30 cycles of 94°C for 45 sec, 52°C for 30 sec, and 72°C for 90 sec. A final extension of 10 min at 72°C is added at the end of the program to ensure complete amplification of the target region.</p>
<p> </p>
<ol start="3">
<li><strong> Check for PCR product on a gel</strong></li>
</ol>
<p> </p>
<ol start="4">
<li><strong> Amplicon Cleaning</strong></li>
</ol>
<ul>
<li>Combine the triplicate PCR reactions into a single volume</li>
<li>Clean PCR reactions with a PCR clean up kit. The MoBio UltraClean PCR clean up kit and the Invitrogen Purelink PCR purification kit have both worked well.</li>
</ul>
<p> </p>
<p> </p>
<ol start="5">
<li><strong> Amplicon Quantification</strong></li>
</ol>
<ul>
<li>Use the qubit system for quantifying DNA, with a few modifications.</li>
<li>Instead of using the qubit assay tubes, prepare the samples in black microplates.</li>
<li>Perform assay with 1 ul of PCR product</li>
<li>Read the plates on the microplate reader in the Allison lab set at 485ex /530em. Estimate amplicon concentration using the standard curve.</li>
</ul>
<p> </p>
<p> </p>
<ol start="6">
<li><strong> Amplicon Pooling</strong></li>
</ol>
<ul>
<li>Combine equal amounts of DNA per sample into single vessel (usually a 15 mL tube).</li>
<li>Concentrate pooled amplicons using the Invitorgen Purelink PCR purification kit</li>
<li>Run a gel to visualize pooled samples</li>
<li>Depending on how your pooled amplicons look on the gel, you may need to gel purify the pooled amplicons. Pyrosequencing is biased towards shorter reads. If you have a messy band on your gel, it is important to purify your samples to improve the quality of the DNA you will receive.
<ul>
<li>We used the Qiagen QIAquick Gel Extraction Kit. We ran samples out on a 1.4 % gel.</li>
<li>The main drawback to performing a gel extraction is that you will almost certainly lose some of your sample. You may need to pool additional PCR product and perform a second gel extraction to obtain enough sample for sequencing.</li>
<li>Remove contaminants from gel purified samples using an Invitrogen Purelink PCR purification kit.</li>
</ul>
</li>
</ul>
<p> </p>
<p> </p>
<ol start="7">
<li><strong> Estimate final sample concentration and purity</strong></li>
</ol>
<ul>
<li>Use the qubit system for estimating sample concentration. The sequencing facility requires 2-3 ug of DNA for sequencing.</li>
<li>Use the Nanodrop in the Martiny lab for estimating sample purity. Samples with an A260/A280 ratio of ~1.8 typically provide optimal sequencing results.</li>
</ul>
<p> </p>
<p> </p>
<ol start="8">
<li><strong> Send sample for sequencing</strong></li>
</ol>
<ul>
<li>Send samples to the Engencore sequencing facility at the University of South Carolina. Check their website for more specific details about what else to include in the shipment.</li>
<li>Send samples overnight on dry ice</li>
</ul>

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