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Star Republic: Guide for Biologists

Purification --- PAGE

Edited by Chang Zhu
1. Cast a 15-20% PAGE/8M Urea gel with wide slot comb.

2. Dissolve dried oligo in loading buffer (9:1 formamide/1X TBE). Concentration should be 30-60 mg/ml (1-2 ODU).

3. Load the sample. And load a loading buffer with dyes on the out wells (loading buffer + 0.01% bromophenol blue, 0.01% xylene cyanol FF).

4. Run until bromphenol blue dye reaches the bottom.

5. Wrap the gel in plastic. Visualize the band under UV shadowing.

6. Cut out the oligo band using a clean razor blade. Cut slightly to the interior of the band to exclude sequences that are not full-length.

7. Crush the gel and transfer to a 15 ml tube.

8. Add 5 ml 0.5 M NaCl, 0.1 M Tris-HCl (pH 8.0), 1 mM EDTA.

9. Shake at room temperature overnight.

10. Centrifuge to pellet down the acrylamide.

11. Extract with 5 ml of phenol/chloroform/isoamyl alchohol (25:24:1).

12. Precipitate with 1 volume of isopropanol.

13. Alternatively, the oligos can be desalted using a desalting column.