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=X3 Dilution Test ([[Daniel:Notebook/ComboLock/2016-8-29|Monday]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] After talking with Dr. Zhang today, I'm going to sequence the bad products from [[Daniel:Notebook/ComboLock/2016-8-30|Tuesday]]. This will hopefully elucidate the issues with the unwanted products, and can help in primer design for reducing the "noise" associated with amplifying these off-target products. ==Protocol== <ol start="5"> <li>Size Select Gel</li> <ol type="A"> <li>For sample lanes, mix 10 uL each sample from lanes 1A-H and lane A12 (mix in one tube), 20 uL 6X loading dye and 10 uL TBE (120 uL total)</li> <li>For ladder lanes, mix 2 uL 25bp ladder, 5 uL 6X loading dye and 23 uL TBE (30 uL total)</li> <li>Add 30 uL to each lane (2 gels total, 1 per sample)</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 bands at 104 bp and put into tubes; 2 lanes per tube (4 tubes total, 2 per sample)</li> <li>Image in gel doc post-extraction</li> <gallery perrow=2 heights=300px widths=300px> File:2016-09-02-X3Dilution-SizeSelect.png|Before Image File:2016-09-02-X3Dilution-SizeSelect-After.png|After Image </gallery> <li>Centrifuge tubes at 12000rpm 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 12000 rpm for 1.5 minutes</li> <li>Extract the supernatant and place into NanoSep column</li> <li>Centrifuge at 12000 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/2016-9-3|tomorrow]]</li> [[Category:ComboLock]] [[Category:20160829]]
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