Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Summary

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

Abstract

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method¹. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 °C during the gel run allows for the separation of unstructured DNA or RNA molecules.

In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.

In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol ^1,2.

Protocol

The complete and detailed text protocol for this experimental procedure is available in Current Protocols in Molecular Biology. Detailed step-by-step instructions for the assembly of the gel sandwich and for gel apparatus can be found on the BioRad website ³.

Required equipment:
Glass plates (inner and outer)
10 cm cell: 10.1 x 7.3 cm (inner plate), 10.1 x 8.2 cm (outer plate), BioRad
20 cm cell: 20 x 20 cm (inner plate), 20 x 22.3 cm (outer plate), BioRad
0.5-1.5 mm gel comb and spacers
Gel casting stand
Gel apparatus with lid and cables
High voltage power supply
H....

Discussion

1. The band sharpness depends on several factors. The volume of sample loaded for sharp bands should be as small as possible, i.e. ideally between 1-5 μl. However, up to 15 μl can be loaded which still ensures acceptable resolution. Less sample volume results in sharper bands.
2. The band quality is also dependent on the gel thickness. Thinner gels, such as 0.75 mm show better results than 1.5 mm thick gels.
3. The shape of bands can also be influenced during gel loading, if the sample is not applied equally into the gel.......