Gel electrophoresis, electroelution and ethanol precipitation
Gel electrophoresis of the linear DNA library samples were performed on a 2% agarose Tris-Acetate-EDTA (TAE) gel, run at constant 100V. Transcribed mRNA libraries and their FITC-puromycin-FITC DNA tag ligated products (mRNA-tag) were resolved using mini size (8 ml) 8 M urea 6% polyacrylamide Tris-borate-EDTA (TBE) gel at a constant 50 mA. SYBR Gold staining (Thermo Fisher Scientific) was used to detect DNA and mRNA products. For gel excision, ligation product was run on a large-scale urea-TBE gel (40 ml). A clean scalpel was used to cut off a gel strip containing mRNA-tag under a blue light LED transilluminator (FUJIFILM Wako Pure Chemical). Gel purification was performed using standard electroelution method using a Model 422 Electro-Eluter (Bio-Rad). First, the gels were crushed into small pieces and placed inside the Electro-Eluter tubes (Bio-Rad) with membrane cap (12 kDa cut-off) attached. Tubes were filled with 1x TBE buffer and run at 10 mA/tube for 30 min. Then, the polarity was reversed for 1 min to release the mRNA-tag from the membrane. Eluted sample solution was recovered and subjected to ethanol precipitation. For ethanol precipitation, 10% sample volume of 3 M sodium acetate was added to the eluted sample solution, then 3 volumes of 99.5% ethanol (FUJIFILM Wako Pure Chemical) was added. After mixing, the was incubated overnight at -20 ℃ to help increase the yield, and the mixture then centrifuged at 4℃, 20,000 x g for 60 min. The supernatant was carefully discarded, and 1 ml of 70% ethanol was added to the white pellet. A second centrifugation was performed at 4 ℃, 20,000 x g for 15 min. The supernatant was again carefully discarded, and the pellet was air-dried at room temperature for 30 min. Finally, the pellet was resuspended in RNase free water and quantified by a Nanodrop 2000c.