Protocol for PAGE Gel Purification
(Information taken from ABI "Evaluation and Isolating Synthetic Oligonucleotides")
- Desalt long (over 50 nucleotides) oligos before electrophoresis.
dried oligo in loading media (9:1 (v:v) formamide/1X TBE) to a concentration
1-2 ODU per µL.
- If any samples do not readily dissolve, heat to 60C
- Do NOT load marker dyes with the oligos. Load bromophenol
blue and xylene cyanol in
- Different gel concentrations should be used for different length oligos.
of the gel and width of the comb teeth determine the well surface
the amount of crude oligo that can be loaded. For example, a 1.5mm
gel and comb teeth
2-3cm wide allows for 10-12 ODU (A260) of oligo.
- Thick gels require higher
power settings (30-50W) and/or longer run times.
- The oligo should travel
at least 2/3 the length of the gel but the further the oligo
travels the better the seperation from failure sequences.
Visualization and Recovery
- Pry apart the glass plates and place the gel on a plastic wrapped flourescent
- By holding a shortwave UV light source (approx. 240nm) directly
above the oligo (so that
the shadow does not appear slightly different from the acutal oligo),
the varying bands
can be seen.
- Be careful not to expose the oligo to UV light longer than neccesary
because it can cause
- If the oligo is not degenerate, using a clean
razor blade, cut out the band by cutting
slightly to the interior of the band to eliminate all n-1 sequences.
the oligo is degenerate cuts should be made slightly outside the
- Place the excised band in a tube or fritted column.
- Recover product from
gel by soaking the gel slice in 1mL of any of the following:
NaCl, 2M triethylammonium acetate
- 50mM triethylammonium acetate
- 0.5M NaCl, 0.1M Tris-HCl (pH 7.0)
containing 1mM EDTA
- Deionized water
1. Incubate gel slice at room temperature for at least 12 hours.
2. Decant and save the solution.
3. To remove urea, salts, and gel debris, desalt the oligo.
Recovery yields are from 10-80% of crude product.