TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Extraction of high quality DNA for long read sequencing e.g. PacBio</p>
<p>Optimized for DNA extraction from wheat stripe rust spores and also tested on barley leaf rust.</p>
<p>Buffers are best when fresh and not older than 3-6 months. Buffered Phenol:Chloroform:Isoamylalcohol (25:24:1) should not be older than 3 months.</p>
<p>Critical steps to obtain high quality DNA:</p>
<ul>
<li>Do NOT heat samples during DNA extractions! Perform all steps at RT or 4oC as indicated.</li>
<li>Do NOT incubate samples with KAc for prolonged time periods</li>
<li>Perform two steps of buffered Phenol:Chloroform:Isoamylalcohol purification to reduce co-purifying metabolites.</li>
</ul>
<p> </p>
<p>DNA fragments were well above the 40kb mark based on Pippin Pulse Gels. The sequencing center performed a second AMPure purification step before library construction. Summary statistics of sequencing runs to follow.</p>

[GUIDELINES]
<p>Modified from protocols of Prof. Pietro Spanu (Imperial College, London) and T. M.</p>
<p>Fulton, J. Chunwongse, S. D. Tanksley, Pl Mol Biol Rep 13, 207 (1995)<br /><br />I am gratefull for critical suggestion from the following scientists.<br />Dr. Claire Anderson, Dr. Andril Gryganskyi, and Dr. David Hayward.<br /><br /></p>
<p>Optimized for DNA extraction from wheat stripe rust spores and also tested on barley leaf rust</p>
<p><strong>Buffers are best when fresh and not older than 3-6 months. Buffered Phenol:Chloroform:Isoamylalcohol (25:24:1) should not be older than 3 months.</strong></p>
<p> </p>
<p>Critical steps to obtain high quality DNA:</p>
<ul>
<li>Do NOT heat samples during DNA extractions! Perform all steps at RT or 4oC as indicated.</li>
<li>Do NOT incubate samples with KAc for prolonged time periods</li>
<li>Perform two steps of buffered Phenol:Chloroform:Isoamylalcohol purification to reduce co-purifying metabolites.</li>
</ul>
<br />
<p><strong>Reagents required</strong></p>
<p><strong>BUFFER A:</strong> 0.35 M sorbitol</p>
<p>0.1 M Tris-HCl, pH 9</p>
<p>5 mM EDTA, pH 8</p>
<p>autoclave to sterilize</p>
<p> </p>
<p><strong>BUFFER B:</strong> 0.2 M Tris-HCl, pH 9</p>
<p>50 mM EDTA, pH 8</p>
<p>2 M NaCl</p>
<p>2% CTAB</p>
<p>autoclave to sterilize</p>
<p> </p>
<p><strong>BUFFER C:</strong> 5% Sarkosyl (N-lauroylsarcosine sodium salt SIGMA L5125)</p>
<p>Filter-sterilize</p>
<p> </p>
<p><strong>Other solutions</strong>:</p>
<p>Potassium Acetate 5M (KAc precipitate polysaccharides) pH 7.5</p>
<p>Polyvinylpyrrolidone (40000 MW) 1 % [w/v] (Sigma PVP40)</p>
<p>Sodium Acetate (NaAc) 3M pH 5.2</p>
<p>Filter-sterilize</p>
<p> </p>
<p>Isopropanol 100%</p>
<p>Ethanol 70%</p>
<p>Buffered Phenol:Chloroforme:Isoamylalcool P:C:I (25:24:1, Sigma P2069)</p>
<p>Autoclave acid washed Sand</p>
<p> </p>
<p><strong>Enzymes</strong></p>
<p>RNAse T1 (1000 U/ml, Thermo Fisher EN0541)</p>
<p>Proteinase K (800U/ml, NEB P81072)</p>
<p> <br />AMPure beads from Beckman</p>
<p><br />Lysis Buffer For 17.5 ml for 500 mg starting material</p>
<p>2.5 volume of Buffer A 6.5 ml</p>
<p>2.5 volume of Buffer B 6.5 ml</p>
<p>1.0 volume of Buffer C 2.75 ml</p>
<p>PVP 0.1 % 1.75 ml</p>
<p> </p>

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