Denaturing Urea Polyacrylamide Gel Electrophoresis for RNA Analysis: A Technique to Separate Fluorescently Labeled Phosphorylated RNA Oligonucleotides from their Non-Phosphorylated Equivalents
Transcript
To prepare a 15% denaturing acrylamide gel solution, combine 22.5 milliliters of pre-mixed 40% 29:1 acrylamide/bis-acrylamide solution, 6 milliliters of 10X TBE, 28.8 grams of urea, and RNase-free water to a total volume of 59 milliliters. Then, gently stir the solution.
Heat the solution in the microwave for 20 seconds, stir it, and immediately return it to the microwave for another 20 seconds. Gently stir the solution until the urea is completely dissolved. Place the glass beaker into a shallow water bath, containing cold water, for 5 minutes, making sure that the level of cold water surrounding the glass beaker is above the level of solution inside the glass beaker. When the solution is cool, filter and de-gas it with a 0.22-micrometer disposable filtration unit to remove particulates and microscopic air bubbles.
Wash a short and long glass plate with soap and water. Then, spray each plate with 95% ethanol, and wipe the glass to remove any moisture. Elevate the long plate off the bench top by placing it on top of a box. Then, position 0.4-millimeter spacers along the long edges of the plate. Lay the short plate on top of the long plate, making sure that the edges of the short plate, long plate, and spacers are aligned. Then, clamp each side with three evenly-spaced metal clamps.
Add 24 microliters of TEMED to the acrylamide solution and mix it. Then, add 600 microliters of 10% APS, and immediately pour the solution between the glass plates.
Pouring the acrylamide solution between the glass plate sandwich can be really challenging. To avoid air bubbles, tap the glass plate sandwich while you're pouring your solution.
Carefully, add a clean 32-well comb to the top of the glass plate sandwich, and allow the acrylamide to polymerize for 30 minutes.
To run the gel, set the heat block to 75 degrees Celsius, remove the metal clamps, and thoroughly wash and dry the glass plate sandwich. Position the plate sandwich in the gel apparatus with the short plate facing forward, and prepare 0.5X TBE running buffer by combining 100 milliliters of 10X TBE with 1.9 liters of RNase-free water.
Add 600 milliliters of the running buffer to the upper and lower chambers of the apparatus. Gently remove the comb from the gel and thoroughly rinse the wells with a syringe. Pre-run the gel at 50 watts for 30 minutes. Then, rinse the wells again.
Pulse spin the quenched reactions, and incubate them at 75 degrees Celsius for 3 minutes. Repeat the pulse spin, and immediately load 10 microliters of each sample onto the gel. Then, run the gel for 3 hours at 50 watts. When the run has finished, turn off the power supply and drain the upper chamber of the apparatus.
Wash and dry the outer side of the glass plate sandwich. Then, cover it with foil and transfer it to a laser scanner for imaging. Mount the glass plate sandwich onto the stage of the laser scanner, set the excitation and emission wavelengths for the desired fluorophore, and image the gel.
