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[DESCRIPTION]
<p><strong>SpinSmart Plasmid Miniprep kits</strong> are designed to rapidly purify plasmid DNA from bacterial cultures.</p>
<p> </p>
<p>The protocol below is appropriate for both <a href="https://www.denvillescientific.com/products/spinsmart%E2%84%A2-plasmid-miniprep-dna-purification-kit-with-reagents--50-preps" target="_blank">CM-410-50</a> and <a href="https://www.denvillescientific.com/products/spinsmart%E2%84%A2-plasmid-miniprep-dna-purification-kit-with-reagents--250-preps" target="_blank">CM-410-250</a>.</p>

[BEFORE_START]
<p>Prior to starting the SpinSmart Plasmid Miniprep prepare the following:</p>
<p> </p>
<p>Before the first use of the kit, add 1 ml of PB1 Resuspension Buffer to the RNase A vial and vortex briefly. Transfer the mixture into the PB1 bottle and mix thoroughly. <br />Store PB1 Resuspension Buffer (containing RNase A) at 4°C for up to 6 months.</p>
<p> </p>
<p>Add the indicated volume of 96-100% ethanol to PB5 Wash Buffer.</p>
<p> </p>
<table style="width: 515px;">
<tbody>
<tr>
<td style="width: 140px;"><strong>Catalog Number</strong></td>
<td style="width: 173px; text-align: center;">
<p><strong>50 preps</strong></p>
<p><strong>Cat. No. CM-410-50</strong></p>
</td>
<td style="width: 184px; text-align: center;">
<p><strong>250 preps</strong></p>
<p><strong>Cat. No. CM-410-250</strong></p>
</td>
</tr>
<tr>
<td style="width: 140px;">Buffer PB5 (Concentrate)</td>
<td style="text-align: center; width: 173px;">2 x 6 ml<br />add 24 ml 96% - 100%<br />EtOH</td>
<td style="text-align: center; width: 184px;">2 x 20 ml<br />add 80 ml 96% - 100%<br />EtOH</td>
</tr>
</tbody>
</table>
<p> </p>

[GUIDELINES]
<p><strong>Spin Smart Plasmid Miniprep Kit Components </strong></p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/g76cz36.png" data-src="https://s3.amazonaws.com/pr-journal/g76cz36.png" data-ofn="Screen Shot 2016-12-13 at 12.48.42 PM.png" /></p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p><strong>Equipment to be supplied by user </strong> </p>
<p>96-100% ethanol</p>
<p>1.5 ml microcentrifuge tubes</p>
<p>Manual piEquipment to be supplied by userpettors and disposable pipette tips</p>
<p>Centrifuge for microcentrifuge tubes</p>
<p>Personal protection equipment (lab coat, gloves, goggles)<strong> <br /> </strong></p>
<p> </p>
<p><strong>Product description</strong></p>
<p> </p>
<p><strong>SpinSmart Plasmid Miniprep kits</strong> are designed to rapidly purify plasmid DNA from bacterial cultures. Bacteria are grown overnight in culture media, then harvested by centrifugation. Pelleted bacterial cells are resuspended in PB1 buffer, then alkaline lysis buffer PB2 releases plasmid DNA from the cells. The reaction is neutralized by PB3 buffer, which also creates appropriate conditions for binding plasmid DNA to the SpinSmart Plasmid Miniprep silica membrane. A centrifugation step pellets precipitated protein, genomic DNA, and cell debris. The supernatant is loaded onto a SpinSmart Plasmid Miniprep binding column.</p>
<p> </p>
<p>Salts, metabolites, and soluble cellular debris are removed by wash steps with ethanolic PB5 Wash Buffer. After the wash steps, plasmid DNA is eluted from the column with PB6 Elution Buffer (5 mM Tris/HCl, pH 8.5). If host strains with high levels of nucleases are used, we recommend an additional washing step with pre-warmed PB4 Wash Buffer. The additional wash with PB4 will also increase the reading length of automated fluorescent DNA sequencing reactions.</p>
<p> </p>
<p>SpinSmart Plasmid Columns have a DNA binding capacity of 60 µg.</p>
<p> </p>
<p>SpinSmart purified plasmid DNA is suitable for applications like automated fluorescent DNA sequencing, PCR, or other enzymatic reactions.</p>
<p> </p>
<p> </p>
<p> <img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/g77cz36.png" data-src="https://s3.amazonaws.com/pr-journal/g77cz36.png" data-ofn="Screen Shot 2016-12-13 at 12.50.57 PM.png" /></p>
<p> </p>
<p><strong> Growth of bacterial cultures</strong></p>
<p> </p>
<p>Plasmid DNA quality and yield is highly dependent on the type of culture media and antibiotics used, the bacterial host strain, the plasmid type, size, and copy number.  </p>
<p> </p>
<p><strong>LB (Luria Bertani)</strong> medium is recommended for standard, high-copy plasmids. The cell culture should be incubated at 37°C with constant shaking (200-250 rpm) preferably 12-16 h overnight. The culture should be grown to an OD600 of 3-6. Rich media, such as 2xYT (Yeast/Tryptone), TB (Terrific Broth) or CircleGrow, can also be used. Bacteria grow faster in rich media, so they reach the stationary phase much earlier than in LB medium (≤ 12 h), and they also achieve higher cell masses. However, this does not necessarily yield more plasmid DNA. Overgrowing a culture could lead to a high percentage of dead or starving cells, which can result in plasmid DNA that is partially degraded or contaminated with chromosomal DNA. Culture medium and incubation time must be optimized for each host strain/plasmid construct combination individually.</p>
<p> </p>
<p>Cell cultures must be grown under <strong>antibiotic selection</strong> to ensure plasmid propagation. <br />  </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/g78cz36.png" data-src="https://s3.amazonaws.com/pr-journal/g78cz36.png" data-ofn="Screen Shot 2016-12-13 at 12.51.54 PM.png" /></p>
<p>  </p>
<p>∗ Maniatis T, Fritsch EF, Sambrook J: Molecular cloning. A laboratory manual, Cold Spring Harbor, Cold Spring, New York 1982. </p>
<p> </p>
<p><strong>SpinSmart Plasmid Minipreps</strong> recommend to use 5 ml of a well grown bacterial culture, OD<sub>600</sub> = 3.</p>
<p> </p>
<p>Culture volumes can be increased if the cell culture grows very poorly or has to be decreased. Please note the OD<sub>600</sub> values and corresponding culture values below:</p>
<p> </p>
<table style="width: 407px;">
<tbody>
<tr style="height: 20px;">
<td style="width: 479px; height: 20px;" colspan="7"><strong>Recommended Culture Volumes</strong></td>
</tr>
<tr style="height: 20px;">
<td style="width: 145px; height: 20px;">OD<sub>600</sub></td>
<td style="width: 57px; height: 20px;">1</td>
<td style="width: 56px; height: 20px;">2</td>
<td style="width: 57px; height: 20px;">3</td>
<td style="width: 51px; height: 20px;">4</td>
<td style="width: 52px; height: 20px;">5</td>
<td style="width: 61px; height: 20px;">6</td>
</tr>
<tr style="height: 20px;">
<td style="width: 145px; height: 20px;">Culture Volume</td>
<td style="width: 57px; height: 20px;">15 ml</td>
<td style="width: 56px; height: 20px;">8 ml</td>
<td style="width: 57px; height: 20px;"><strong>5 ml</strong></td>
<td style="width: 51px; height: 20px;">4 ml</td>
<td style="width: 52px; height: 20px;">3 ml</td>
<td style="width: 61px; height: 20px;">2 ml</td>
</tr>
</tbody>
</table>
<p> </p>
<p><strong>Elution procedures</strong></p>
<p> </p>
<p><strong>High yield: important for larger constructs:</strong> Heat elution buffer to 70°C, add 50-100 µl to the SpinSmart Plasmid miniprep column.  Incubate at 70°C for 2 min.</p>
<p> </p>
<p><strong> High yield</strong>: Perform two elution steps with the volume indicated in the individual protocol. 90-100% of bound plasmid DNA can be eluted.</p>
<p> </p>
<p><strong>High concentration</strong>: Perform one elution step with 60% of the volume indicated in the individual protocol.  </p>
<p> </p>
<p><strong>High yield and high concentration</strong>: Apply half of the volume of elution buffer as indicated in the individual protocol, incubate for 3 min and centrifuge. Apply a second aliquot of elution buffer, incubate and centrifuge again.  </p>
<p> </p>
<p>SpinSmart Plasmid Miniprep protocols recommend PB6 Elution Buffer (5 mM Tris, pH 8.5) for elution.  We do not recommend buffers containing EDTA, especially if the plasmid DNA is intended for sequencing reactions. Water may be used for elution, however the pH should be adjusted to pH 7.0-8.5 since deionized water typically has a low pH.  </p>
<p> </p>
<p> </p>
<p><strong>Storage and preparation of solutions</strong></p>
<p> </p>
<p>Buffers PB3 and PB4 contain guanidine hydrochloride! Wear gloves and goggles when using this kit!</p>
<p> </p>
<p>All kit components can be stored at room temperature (20-25°C) and are stable for up to one year.</p>
<p> </p>
<p>Keep buffer bottles tightly closed, especially if buffers are warmed at any time.</p>
<p> </p>
<p>Sodium dodecyl sulfate (SDS) in PB2 Lysis Buffer may precipitate if stored at temperatures below 20°C. If a precipitate is observed, incubate the bottle at 30–40°C for several minutes and mix well.</p>
<p> </p>
<p>Prior to starting the SpinSmart Plasmid Miniprep prepare the following:</p>
<p> </p>
<p>Before the first use of the kit, add 1 ml of PB1 Resuspension Buffer to the RNase A vial and vortex briefly. Transfer the mixture into the PB1 bottle and mix thoroughly. <br /> Store PB1 Resuspension Buffer (containing RNase A) at 4°C for up to 6 months.</p>
<p> </p>
<p>Add the indicated volume of 96-100% ethanol to PB5 Wash Buffer.</p>
<table style="width: 515px;">
<tbody>
<tr>
<td style="width: 140px;"><strong>Catalog Number</strong></td>
<td style="width: 173px; text-align: center;">
<p><strong>50 preps</strong></p>
<p><strong>Cat. No. CM-410-50</strong></p>
</td>
<td style="width: 184px; text-align: center;">
<p><strong>250 preps</strong></p>
<p><strong>Cat. No. CM-410-250</strong></p>
</td>
</tr>
<tr>
<td style="width: 140px;">Buffer PB5 (Concentrate)</td>
<td style="text-align: center; width: 173px;">2 x 6 ml<br />add 24 ml 96% - 100%<br />EtOH</td>
<td style="text-align: center; width: 184px;">2 x 20 ml<br />add 80 ml 96% - 100%<br />EtOH</td>
</tr>
</tbody>
</table>
<p><br /><br /></p>
<p> </p>
<p><strong>Safety Information</strong></p>
<p><em>Wear gloves and goggles and follow the safety instructions given in this section.</em></p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/g79cz36.png" data-src="https://s3.amazonaws.com/pr-journal/g79cz36.png" data-ofn="Screen Shot 2016-12-13 at 1.02.41 PM.png" /></p>
<p> </p>
<p>                                               <strong>  Risk Phrases</strong> <br />R 10 Flammable</p>
<p>R 22 Harmful if swallowed</p>
<p>R 36/38 Irritating to eyes and skin</p>
<p>R 42/43 May cause sensitization by inhalation and skin contact <br /> <br />                                                         <strong>  Safety Phrases</strong></p>
<p>S 7 Keep container tightly closed</p>
<p>S 16 Keep away from sources of ignition - No Smoking!</p>
<p>S 22 Do not breathe dust</p>
<p>S 24 Avoid contact with the skin</p>
<p>S 25 Avoid contact with the eyes</p>
<p>S 26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice</p>
<p>S 37/39 Wear suitable protective clothing and gloves</p>
<p>S 45 In case of accident or if you feel unwell, seek medical advice immediately</p>
<p>         (show the label where possible) <br /> <br /> <br />∗ Label not necessary, if quantity below 25 g or ml (concerning 67/548/EEC Art. 25, 1999/45/EC Art. 12 and German GefStoffV § 42 and TRGS 200 7.1)</p>
<p>∗∗ Label not necessary, if quantity below 125 g or ml (concerning 67/548/EEC Art. 25, 1999/45/EC Art. 12 and German GefStoffV § 42 and TRGS 200 7.1)</p>
<p> </p>
<p><strong>Troubleshooting</strong></p>
<p>Please see the product manual for troubleshooting information.</p>
<p><a href="https://www.denvillescientific.com/Files/Files/Denville/ProductDocs/CM-410-50_and_M-410-250_manual.pdf" target="_blank">https://www.denvillescientific.com/Files/Files/Denville/ProductDocs/CM-410-50_and_M-410-250_manual.pdf</a></p>
<p> </p>
<p> </p>

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