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 crosslink 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:
1. Bromophenol blue (BPB), in a 15% gel this dye runs as a 12mer; in a 20% gel this runs as an 8mer
2. 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
e 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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