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=X3 Dilution Test (Started [[Daniel:Notebook/ComboLock/2016-8-29|Monday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Protocol== <ol start="5"> <li>Size Select Gel</li> <ol type="A"> <li>For sample lanes <ol type="a"> <li>Mix 10 uL from each replicate (30 total) in separate tubes for each sample</li> <li>Use lanes 1A-H and lane A12</li> <li>Add 6 uL 6X loading dye and 4 uL TBE for 40 uL total</li> <li>For sample 1, do this twice (80 uL total)</li> </ol> <li>For ladder lane mix 4 uL 25bp ladder, 6 uL 6X loading dye and 30 uL TBE (40 uL total)</li> <li>Add 40 uL to each lane</li> <li>Run gel for 25 minutes at 250V</li> <li>While running, prepare tubes by puncturing a 0.5 mL tube with a 0.22 gauge needle and putting inside a 1.5 mL tube</li> <li>Stain gel for 3 minutes with 3 uL SYBR gold</li> <li>Image in gel doc</li> <li>Extract desired bands (see gel image) and put into tubes; 2 lanes per tube (2 tubes total)</li> <li>Image in gel doc post-extraction</li> <gallery perrow=2 heights=300px widths=300px> File:2016-09-05-X3Dilution-SizeSelect.png|Before Image File:2016-09-05-X3Dilution-SizeSelect-After.png|After Image </gallery> <li>Centrifuge tubes at 14000rpm for 1.5 minutes</li> <li>Discard 0.5 mL tube and resuspend in 500 uL TE buffer</li> <li>Incubate at 37C for 1 hour with vigorous shaker</li> <li>Centrifuge at 14000 rpm for 1.5 minutes</li> <li>Extract the supernatant and place into NanoSep column</li> <li>Centrifuge at 14000 rpm for 1.5 minutes</li> </ol> <li>Ethanol Precipitation</li> <ol type="A"> <li>In a 2 mL tube, add 1250 uL 100% EtOH, 50 uL 3 M NaOAc, and 2 uL glycoblue to 500 uL sample</li> <li>Incubate at -80C overnight; continued [[Daniel:Notebook/ComboLock/2016-9-6|tomorrow]]</li> [[Category:ComboLock]] [[Category:20160829]]
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