Charles Zuker

PURIFICATION OF OLIGONUCLEOTIDES BY THIN LAYER CHROMATOGRAPHY (TLC)

For practical purposes TLC-purified oligonucleotides; are as good as gel-of-HPLC-purified oligos. TLC;, however, is much cheaper.

1) Lyophilize oligonucleotide; (100-300µg); resuspend in 100-300µl of distilled water.

2) Load oligo on a Silica Gel TLC plate; (Silica Gel 60 F-254, EM Reagents, Cat. 5765-7, 20x20 cm) as a thin line across the plate approximately 2 cm from the bottom.

3) Insert plate on a chromatography tank (30x30 cm) containing 100 ml running buffer (55ml n-propanol, 35ml NH4OH, 110ml H2O). Run until front is at or near the top.

4) Remove plate and let dry inside a hood for a few hours (or until completely dry). Visualize oligos using short-wave UV lamp and use a razor blade to scrape off your oligonucleotide (usually the lowest and most intense band of the ladder of oligos).

5) Add silica powder to eppendorf tube containing 300-600µl of water, vortex for a few min and spin down for 10 min. Re-extract the silica with 1/3 volume, spin, mix supenatants and respin for 5 min.

6) Read O.D. of your purified oligo (260 nm).