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Dinh:Probes Prep May2015
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===PAGE purification (Day 2/3)=== * Purified A, B * Reagent: 4 TBE-Urea gels, 300 uL TBU loading buffer, 6 Nanosep columns. * We ran out of agarose so I used PAGE instead. * Prepared 2D TBE-Urea gels for loading. Loaded 75 uL of sample + 75 uL of 2x loading buffer into each well. I used 2 gels per sample (22 PCR reactions). * I didn't use a ladder because I already ran these samples with a ladder on the digestion check gel. * Prepared the gel-shearing apparatus (1x2.0 mL tube per 2D gel with 1x0.5 mL inside - pierced the 0.5 mL tube with a 20 gauge needle, cut off the cap of the 2.0 mL tube and save for later) and UV them. * Ran the gels at 220 V for 30 minutes. Stain with Sybr Gold for ~30 seconds. * Wrap each gel flat with plastic wrap and under UV light, use a Sharpie marker to trace the band to be excised on the plastic wrap. * Use a No. 10 scapel to cut out the desired bands and transfer sliced gel into the pierced 0.5 mL tube. Image the gel within the plastic wrap to ensure correct fragment was excised. * Spin the tubes at maximum speed for 2 minutes. * To each tube, at 3X(gel volume) of 1X TE buffer. I used 1.4 mL of TE per gel. Cap & freeze the tubes at -80C * Thaw each tube in the thermomixer for 30 minutes at 50C. Ramp temperature up to 94C and soak for 5 minutes. Ramp temperature down to 37C and soak for 30 minutes. * Separate gel from elutant using 1 Nanosep column (3 rounds centrifugation) per tube of gel. Transfer the elutant to a 5.0 mL tube. * To each 5.0 mL tube with 1.4 mL elutant, add 140 uL 3.0 M sodium acetate, 5 uL GlycoBlue, 3.5 mL of 100% ethanol. Cap & freeze each tube at -80C. * Use the ultracentrifuge in the basement to (SORVALL) to spin down each 5.0 mL tubes at 12,000rpm for 20 minutes. * Transfer the pellet to a 1.5 mL tube with ice cold 75% ethanol & spin down & dry. After drying dissolve each pellet with 50 uL of water. {| class="wikitable" | align="center" style="background:#f0f0f0;"|'''Subset''' | align="center" style="background:#f0f0f0;"|'''Total QUBIT (ug)''' | align="center" style="background:#f0f0f0;"|'''PAGE/EtohPPT (ng/uL)''' | align="center" style="background:#f0f0f0;"|'''Volume (uL)''' | align="center" style="background:#f0f0f0;"|'''Yield QUBIT (ug)''' | align="center" style="background:#f0f0f0;"|'''%yield''' |- | A||3.1784||8.12||50||0.406||12.77% |- | B||2.5272||7.72||50||0.386||15.27% |}
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