Denaturing 7M Urea PAGE Gels
For Nucleic Acid Purification
Polyacrylamide gel electrophoresis (PAGE) remains one of the best tools for the purification of oligos and nucleic acids in general (RNA and other second/third generation molecules, e.g. LNA’s, UNA’s, etc).
The 7M urea ensures the linearity of the nucleic acids, by avoiding secondary/tertiary structure formation. 0.1% APS (Ammonium persulfate) and 0.1%TEMED (Tetramethylethylenediamine) are used to cross link the polyacrylamide.
Gel percentage. For the separation of oligos up to 30-40 bases a 20% gel is used. For oligos longer than 40 bases a 15% PAGE gel is suggested
Dyes used in PAGE purification. These two dyes have proven very useful:
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Bromophenol blue (BPB), in a 15% gel this dye runs as a 12mer; in a 20% gel this runs as an 8mer
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Xylene Cyanol (XC), in a 15% gel this dye runs as a 35mer; in a 20% this dye runs as a 24 mer
Therefore, user is advised to consider these mobilities when applying oligos in the PAGE gel; if you have a series of 24mers and you are running a 20% gel, you may want to only use BPB as your reference dye and skip XC, as this will overlap your 24mer oligos.
Analytical PAGE gel. For quality control purposes a 0.5-1.0 mm thickness comb/spacer combination is suggested. Need about 0.5 OD of the oligo in order to visualize it by UV shadowing.
Preparative PAGE gel. A comb/spacer combination with 3-5mm thickness is suggested; depending on the length of the well, user can purify 10-20 OD’s oligos per well (1x1/4 inch well).
Base composition effect. Two oligos of the same size but different base compositions, will not run at same distance in a PAGE gel. More on this subject on a separate report.
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