Rna ligase buffer

RNA samples and markers were denatured by diluting 1:1 volumetrically with the indicated sample buffers for each figure. In all cases except for Northern blots, the 2x sample buffers contained 95% formamide. For EDTA testing, sample buffers were prepared with 95% formamide and either 18mM or 50mM EDTA (Figure 3B and 3C). For SDS testing, 0.025% SDS was included with 95% formamide. For SYBR-GOLD testing, 0.025% SYBR-GOLD was added to 95% formamide. Commercially available 2x RNA sample buffers were used where indicated (GLB II: Gel Loading Buffer II, ThermoFisher; RLD: RNA Loading Dye 2X, ThermoFisher). For carryover buffer testing, 1x RNA ligase buffer (New England Biolabs) was added to the sample prior to heating. In all cases, samples were volumetrically diluted 1:1 to a final volume of 20ul/well, heated to 70°C for at least 2 minutes, and chilled on ice for at least three minutes. Samples were loaded onto either 2% E-Gel^™ EX Agarose Gels with SYBR-GOLD II^™ or 2% E-Gel^™ Agarose Gels with SYBR^™ Safe and run on the E-Gel^™ Powersnap Electrophoresis System using programs “EX1–2%” or “SizeSelect2%” for the times indicated in the figures at room temperature. Images were taken with a Bio-Rad Gel Doc^™ XR and Image Lab 5.2 software using the “SYBR-Safe” settings. For Northern analysis, the E-Gel^™ cassettes were opened and carefully placed onto Brightstar nylon membranes.