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.
Rna loading dye 2x
Manufactured by Thermo Fisher Scientific
RNA Loading Dye 2X is a concentrated solution used to prepare RNA samples for gel electrophoresis. It contains dyes that allow the visualization of RNA bands during the electrophoresis process.
Automatically generated - may contain errors
Lab products found in correlation
Gel loading buffer 2, by Thermo Fisher Scientific (1 mentions)
Image lab 5, by Bio-Rad (1 mentions)
Rna ligase buffer, by New England Biolabs (1 mentions)
Gel doc xr, by Bio-Rad (1 mentions)
Rneasy mini isolation kit, by Qiagen (1 mentions)
Agarose, by NZYTech (1 mentions)
Sybr gold, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
3 protocols using rna loading dye 2x
1
RNA Sample Preparation for Electrophoresis
2
Extraction and Analysis of rRNA
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In the experiment where we analyzed the rRNA content in fractions of the sucrose gradients (Figure 1A ), fractions containing the free 30S, free 50S and 70S ribosomes were collected independently. Fractions for each subunit were pooled together and the ribosomal particles pelleted overnight at 120,000g. Pellets were resuspended in 80 μl of water, and the concentration was determined. Volume was adjusted to 100 μl and we ensured that the total amount of rRNA did not exceed 100 μg. Proteinase K was added to a final concentration of 100 μg/ml and the mixture was incubated at room temperature for 30 min. We then added 350 μl of RLT buffer from Qiagen RNeasy mini isolation kit and 250 μl of 100% ethanol. The reaction was mixed and loaded on the column provided by the Qiagen RNeasy mini isolation kit. Subsequent washes of the column were done according to the manufacture's protocol. RNA Loading Dye 2X (ThermoScientific) was added to 2 μg of purified rRNA samples, heated at 70ºC for 10 min and put on ice for 5 min before loading into a modified agarose gel containing 0.7% agarose and 0.9% Synergel (Diversified Biotech) in 0.5× TBE buffer. RNA was separated by electrophoresis and visualized under UV light.
3
Polyplex Destabilization by Heparin
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Purified polymer candidates were complexed with 250 ng Cre mRNA at the optimized mRNA:polymer mass ratios for 10 min. Polyplexes were then incubated for 30 min at 37 °C with 2.5–50 µg of heparin (Calbiochem), corresponding to heparin:mRNA ratio ranging from 10:1–200:1. Polyplex destabilization in the presence of negatively charged heparin resulted in the release of mRNA, which was measured by agarose gel electrophoresis. Free mRNA was used as a control for electrophoretic migration upon polyplex dissociation. agarose gels were prepared by dissolving 1% (w/v) agarose (ultrapure grade, NZYTech) in TBE 1x and staining the molten gel with 1:10 000 SYBR Gold (Invitrogen) to visualize mRNA bands. Samples were loaded with 5 µL RNA loading dye 2x (Thermo Scientific), totaling a loading volume of 25 µL in each well. Electrophoresis was performed at 100 V for 30 min. Gels were imaged immediately after completing electrophoresis using a ChemiDoc XRS system (BioRad) equipped with a UV light transilluminator. Complexation studies were repeated 2–3 times for each sample.
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