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[DESCRIPTION]
The NEXTflex™ mtDNA-Seq Kit is designed to prepare single or paired-end DNA libraries of mitochondrial DNA (mtDNA) for sequencing using Illumina® platforms. The procedure isolates mitochondrial DNA from genomic DNA (gDNA) by selective digestion of linear nuclear DNA, followed by fragmentation of mtDNA and library preparation. <br /><br />Please see the <a href="http://www.biooscientific.com/Portals/0/Manuals/NGS/5280-01-NEXTflex-mtDNA-Seq-Kit.pdf" target="_blank">full manual</a> for additional details.<br /><br />(Note: This is the procedure for Cell Samples; for Blood Samples, see <a href="https://www.protocols.io/view/NEXTflex-mtDNA-Seq-for-Blood-Samples-dqy5xv" target="_blank">this</a> protocol.)

[BEFORE_START]
Reagent Preparation <br />1. Briefly spin down each component to ensure material has not lodged in the cap or side of tube. Keep on ice and vortex each tube prior to use. <br />2. Allow Agencourt AMPure XP Beads to come to room temperature and vortex the beads until liquid appears homogenous before every use.

[GUIDELINES]
<strong>Contents, Storage and Shelf Life </strong><br />The NEXTflexÃ¢ÂÂ¢ mtDNA-Seq Kit contains enough material to isolate mtDNA and prepare 8 libraries for IlluminaÃÂ® compatible sequencing. The shelf life of all reagents is 12 months when stored properly. 6X Loading Dye, Nuclease-free Water, and Resuspension Buffer can be stored at room temperature. All other components should be safely stored at -20ÃÂ°C.<br /><br /><img class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/xzc3kn.png" alt="" /><br /><img class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/x3c3kn.png" alt="" /><br /><br /><strong>Required Materials Not Provided </strong><br />Ã¢ÂÂ¢ 2 - 8 ÃÂ¼g of genomic DNA in up to 35 ÃÂ¼L nuclease-free water <br />Ã¢ÂÂ¢ (Optional)NEXTflexÃ¢ÂÂ¢ ChIP-Seq Barcodes Ã¢ÂÂ 6 , 12 , 24 , 48 or NEXTflex-96Ã¢ÂÂ¢ ChIP-Seq Barcodes (Cat # 514120, 514121, 514122, 514123, 514124) <br />Ã¢ÂÂ¢ Ethanol 100% (room temperature) <br />Ã¢ÂÂ¢ Covaris System (S2, E210) or other device for fragmenting DNA <br />Ã¢ÂÂ¢ 96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) or similar <br />Ã¢ÂÂ¢ 96 well Library Storage and Pooling Plate (Fisher Scientific, Cat # AB-0765) or similar <br />Ã¢ÂÂ¢ Adhesive PCR Plate Seal (BioRad, Cat # MSB1001) <br />Ã¢ÂÂ¢ Agencourt AMPure XP 5 mL (Beckman Coulter Genomics, Cat # A63880) <br />Ã¢ÂÂ¢ Magnetic Stand-96 (Life Technologies, Cat # AM10027) or similar <br />Ã¢ÂÂ¢ Heat block at 37ÃÂ°C and at 70ÃÂ°C <br />Ã¢ÂÂ¢ Thermocycler <br />Ã¢ÂÂ¢ 2, 10, 20, 200 and 1000 ÃÂ¼L pipettes, multichannel pipettes <br />Ã¢ÂÂ¢ Nuclease-free barrier pipette tips <br />Ã¢ÂÂ¢ Microcentrifuge <br />Ã¢ÂÂ¢ 1.5 mL nuclease-free microcentrifuge tubes <br />Ã¢ÂÂ¢ Low melt agarose such as Low Gelling Temperature Agarose with a melt point of 65ÃÂºC (Boston Bioproducts, Cat # P-730) <br />Ã¢ÂÂ¢ 1X TAE buffer <br />Ã¢ÂÂ¢ SYBR Gold (Invitrogen, Cat # S11494) <br />Ã¢ÂÂ¢ UV transilluminator or gel documentation instrument <br />Ã¢ÂÂ¢ Gel electrophoresis apparatus <br />Ã¢ÂÂ¢ Electrophoresis power supply <br />Ã¢ÂÂ¢ Vortex <br />Ã¢ÂÂ¢ MiVac (SpeedVac)<br /><br /><strong>NEXTflexÃ¢ÂÂ¢ mtDNA-Seq Flow Chart </strong><br />Figure 1: Sample flow chart with approximate times necessary for each step.<img class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/x5c3kn.png" alt="" /><br /><strong>Starting Material </strong><br />The NEXTflexÃ¢ÂÂ¢ mtDNA-Seq Kit has been optimized and validated using 2 Ã¢ÂÂ 8 ÃÂ¼g of genomic DNA isolated either from human blood or cell samples. This kit provides sufficient reagents for 8 nuclear DNA digestions and mtDNA library preparations. <br /><br />It is recommended that the mtDNA isolation test (see "Optional Quality Control of Nuclear DNA Digest" section) is performed with each isolation. Primers and PCR master mix for this test are provided with the kit. Undigested gDNA may be used as a control (not provided). <br /><br /><strong>Reagent Preparation </strong><br />1. Briefly spin down each component to ensure material has not lodged in the cap or side of tube. Keep on ice and vortex each tube prior to use. <br />2. Allow Agencourt AMPure XP Beads to come to room temperature and vortex the beads until liquid appears homogenous before every use. <br /><br />Figure 3A:ÃÂ  Bioanalyzer Gel Image<br /><br /><img class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/yfc3kn.png" alt="" /><br /><br />Figure 3 B: Bioanalyzer Electropherogram Image<img class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/yic3kn.png" alt="" /><br />Figure 3A &amp; 3B: Example of a mtDNA Library Size Distribution. Upon isolation the mtDNA was sheared to 150-200 bp, a total of 18 cycles of PCR were performed. 1 ÃÂ¼L of the resulting library was run on an Agilent High Sensitivity DNA chip to verify size. Using a QubitÃÂ® 2.0 Fluorometer &amp; QubitÃÂ® dsDNA HS Assay Kit, the concentration of the library was determined to be &gt; 10 nM.<br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />

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