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==Protocol-Library Prep== <ol start="6"> <li>Size Selection</li> <ol type="A"> <li>Add 20 uL sample lanes 1 and 8-10, 5 uL 6X loading dye and 5 uL TBE to sample lanes</li> <ol type="a"> <li>Lane 1: X2-1X</li> <li>Lane 8: X3-50X</li> <li>Lane 9: X2-Flip</li> <li>Lane 10:X3-Flip</li></ol> <li>Aliquot 30 uL each into 4 sample lanes</li> <li>2 uL ladder with 5 uL gel loading dye and 28 uL TBE buffer</li> <li>Run gel for 20 min at 250V</li> <li>Stain with 3 uL SYBR Gold for 3 min</li> <li>Image in gel doc; cut out selected bands and place in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube</li> <gallery perrow=2 heights=200px widths=200px> File:2016-08-25-PosConAmpTest2-SizeSelect.png|Before Image File:2016-08-25-PosConAmpTest2-SizeSelect-After.png|After Image </gallery> <li>Centrifuge gel at 14000rpm for 1.5 minutes</li> <li>Remove 0.5 mL tube and add 500 uL TE buffer to shredded gel</li> <li>Incubate at 37C for at least an hour with vigorous shaking</li> <li>Centrifuge at 14000rpm for 1.5 minutes</li> <li>Transfer supernatant to a nanosep column</li> <li>Centrifuge at 14000rpm for 1.5 minutes</li> <li>Transfer flow through to a 2 mL tube</li> </ol> <li>Ethanol Precipitation</li> <ol type="A"> <li>Add 1.25 mL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue to sample</li> <li>Incubate overnight at -80C; continued [[Daniel:Notebook/ComboLock/2016-8-26|tomorrow]]</li></ol> </ol>
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