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[DESCRIPTION]
This is the protocol for QIAgen's QIAprep Spin Miniprep Kit (catalog numbers 27104 or 27106) with their newer version 2.0 spin columns. For more information about the kit, see <a href="https://www.qiagen.com/us/shop/sample-technologies/dna/dna-preparation/qiaprep-spin-miniprep-kit?cmpid=QVenSPP1404quartzySC#orderinginformation" target="_blank">https://www.qiagen.com/us/shop/sample-technologies/dna/dna-preparation/qiaprep-spin-miniprep-kit?cmpid=QVenSPP1404quartzySC#orderinginformation</a>.<br />
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<p>For purification of up to 20 μg molecular biology grade plasmid DNA</p>
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<div id="md_c_main_content_0_content_column_3_Image"><img src="https://www.qiagen.com/%7E/media/nextq/image%20library/s/46/13/s_4613_qiaprep_s/2_9.ashx?h=113&amp;la=en&amp;w=201&amp;hash=879FCB850090F804BE29D2D5D9C0CBBF79A5EC00" alt="QIAprep 2.0 Spin colum Kit" width="201" height="113" /></div>
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<ul>
<li>Ready-to-use plasmid DNA in minutes</li>
<li>Reproducible yields of molecular biology grade plasmid DNA</li>
<li>Single protocol for high- and low-copy vectors</li>
<li>Even higher yields with the <em>High-Yield Supplementary Protocol </em></li>
<li>Improved QIAprep 2.0 Spin Column</li>
<li class="last">GelPilot loading dye for convenient sample analysis</li>
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<div class="copy">The QIAprep Spin Miniprep Kit is designed for isolation of up to 20 μg high-purity plasmid or cosmid DNA for use in routine molecular biology applications, including fluorescent and radioactive sequencing and cloning. Even higher yields (up to 30 μg) can be achieved using the <em>High-Yield <em>Supplementary</em> Protocol</em>.</div>
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[BEFORE_START]
<strong>Growth of bacterial cultures in tubes or flasks</strong><br />Pick a single colony from a freshly streaked selective plate and inoculate a culture of 1–5 ml LB medium containing the appropriate selective antibiotic. Incubate for 12–16 h at 37°C with vigorous shaking. Growth for more than 16 h is not recommended since cells begin to lyse and plasmid yields may be reduced. Use a tube or flask with a volume of at least 4 times the volume of the culture.<br /><br />
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<li>Add the provided RNase A solution to Buffer P1 before use. Use 1 vial RNase A (centrifuge briefly before use) per bottle Buffer P1 for a final concentration of 100 μg/ml. Mix and store at 2–8°C.</li>
<li>Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).</li>
<li>Check Buffers P2 and N3 before use for salt precipitation. Redissolve any precipitate by warming to 37°C. Do not shake Buffer P2 vigorously.</li>
<li>Optional: Add the provided LyseBlue reagent to Buffer P1 and mix before use. Use 1 vial LyseBlue reagent per bottle Buffer P1 for a final dilution of 1:1000 (e.g., 10 μl LyseBlue into 10 ml Buffer P1). LyseBlue provides visual identification of optimum buffer mixing, thereby preventing the common handling errors that lead to inefficient cell lysis and incomplete precipitation of SDS, genomic DNA, and cell debris.</li>
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[GUIDELINES]
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<li>Close the bottle containing Buffer P2 immediately after use to avoid acidification of Buffer P2 from CO2 in the air.</li>
<li>All centrifugation steps are carried out at 13,000 rpm (~17,900 x g) in a<br />conventional, table-top microcentrifuge.</li>
<li>Ensure that the elution buffer is dispensed directly onto the center of the QIAprep membrane for optimal elution of DNA. Average eluate volume is 48 μl from an elution buffer volume of 50 μl (QIAprep spin procedures), and 60 μl from an elution buffer volume of 100 μl (QIAprep multiwell procedures).</li>
<li>For increased DNA yield, use a higher elution-buffer volume.</li>
<li>For increased DNA concentration, use a lower elution-buffer volume</li>
<li>If water is used for elution, make sure that its pH is between 7.0 and 8.5. Elution efficiency is dependent on pH and the maximum elution efficiency is achieved within this range. A pH &lt;7.0 can decrease yield. Note: Store DNA at –20°C when eluted with water, as DNA may degrade in the absence of a buffering agent. DNA can also be eluted in TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.</li>
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[STEPS]
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