Lab Resources

Oligonucleotide Purification

Kati et al, Journal of Biological Chemistry, Vol. 267, No. 36, pp. 25988-97, Dec. 25, 1992

Andrew Ellington and Jack D. Pollard, Jr., Current Protocols in Molecular Biology (1998) 2.12.1-2.12.7

25mer oligonucleotide

5`-GCC TCG CAG CCG TCC AAC CAA CTC A -3`

45mer oligonucleotide

5`-GGA CGG CAT TGG ATC GAG GTT GAG TTG GTT GGA CGG CTG CGA GGC-3`

Order the 25mer and 45mer template from IDT.

Add 1ml ddH2O to each oligo pellet.

Resuspend for longer than 2 hrs on bench top.

Assembled the gel plates: 1.5mm thick spacers and 5-well Hoefer comb.

Let the gel cassette stand in a gel-casting tray.

Pour the following acrylamide mixture into the casting tray to seal the bottom of plates:

Chemical Ingredients Vol.
20% acrylamide/0.8%bis-acrylamide in 1× TBE/7M Urea 80ml
10% APS 1ml
TEMED 200 µl

Tighten the screws as the solution goes up in between the glass plates.

Let the solution polymerize for at least 20 minutes.

Pour the purification gel into the glass plates:

Chemical Ingredients Vol.
20% acrylamide / 0.8%bis-acrylamide in 1x TBE/7M Urea 125ml
1x TBE/7M Urea 125ml
10% APS 1ml
TEMED 200 µl

Add formamide dye (50 µl) to the DNA solution (200µl) and resuspend by heating at 90°C for 5min.

Load 250 µl of DNA/dye into each well.

Run the gel at 100 W until the oligo`s have migrated ½ to ¾ of the way through the gel.

Turn off the power supply. Pry off the top plate.

Cover the gel with plastic wrap and invert the plate onto a TLC plate.

Shine the short-wave (254nm) UV lamp onto the gel.

Cut out the bands and put them into a 15ml conical tube.

Crush the gel with a spatula.

Add 3-5 ml TE buffer, rinsing the spatula at the same time.

Freeze the sample for 30 min at -80°C or until frozen solid.

Quickly thaw in a hot water bath and let soak for 5 min at 90°C.

Elute on a shaker overnight at 37°C.

Centrifuge the tube for 2 min at 1000×g, room temperature, to pellet the gel fragments.

Use a pipette to draw out the buffer into a new 15ml tube.

Filter off any remaining acrylamide fragments by syringe filtering through a 0.2µm disc.

Concentrate the sample by extracting against >1 volume Of N-butanol in the fume hood.

Centrifuge and remove the upper N-butanol layer.

Repeat until the volume of the lower aqueous layer is ~0.5ml.

Transfer the concentrated sample into the a microfuge tube.

Add 3M Na-acetate, pH 5.2, to a final concentration of 0.3M.

Add 2 volumes of 100% EtOH to precipitate DNA.

Chill for 20 min at -20°C.

Pellet the oligonucleotide by centrifuging for 10 min at 12,000×g, 4oC.

Resuspend the pellets in water and determine the concentration on a UV spec.

Contributed by Louise Wang

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