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==Protocol (Continued from [[Daniel:Notebook/PosSequencing/2017-5-20|Saturday May 20]])== <ol start="4"> <li>Dye hybridization</li> <ol type="A"> <li>Mix 20 uL ssDNA (lambda digested) and 20 uL dsDNA (original, non-digested template)</li> <li>Add 1 uL appropriate (10 uM) dye, A488-dc6 for sample 1 and Cy5-dc6 for sample 3</li> <li>Incubate in the dark (RT) for 15 minutes</li> <li>Magnet pulldown, remove supernatant and add 40 uL 2X SSC buffer</li> <li>Add 15 uL to a slide for analysis</li> <li>Seal coverslip with nail polish and wait approximately 30 minutes to dry</li> </ol> <li>Imaging Parameters</li> <ol type="A"> <li>Confocal Microscope</li> <li>488 Laser-3.00%</li> <ol type="a"> <li>PMT Trans-Gain 225V <li>PMT1-Range:500-550-Gain 550V</li> <li>PMT3-Range:750-800-Gain 600V</li></ol> <li>642 Laser-8.00%</li> <ol type="a"> <li>PMT Trans-Gain 300V <li>PMT1-Range:500-550-Gain 6000V</li> <li>PMT3-Range:750-800-Gain 550V</li></ol> </ol></ol>
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