Mark Fortini

GEL PURIFICATION OF OLIGONUCLEOTIDES


 I generally purify 25-mer and larger oligonucleotides; using the following protocol. For smaller oligonucleotides, I use the TLC purification protocol (see follwoing protocol). For many purposes, such as making primers for DNA sequencing,; oligonucleotides may be purified sufficiently by passage through a G-10 spin column; after lyophilization.

1) For gel purification, first dry down unpurified oligonucleotide solution in a SpeedVac or lyophilizer.

2) Redissolve the pellet in 50-100 µl 0.1 N NaOH. Add 1/3 volume of loading buffer (formamide/bromophenol blue dye).

3) Prepare a 12% polyacrylamide gel (20:1 acrylamide:bis) as follows:
-for 250 ml of gel solution (suitable for a 30 cm x 40 cm x 0.75 mm gel)
115 g urea
12.5 ml 20X TBE
63 ml ddH2O
75 ml 40% acrylamide solution (20 acryl:1 bis)
750 µl 25% ammonium persulfate
150 µl TEMED (add just prior to pouring gel)

4) Cast gel using spacers of 0.75 mm thickness and wide-tooth combs (wells should be 2-4 cm wide).

5) Load all or part of each oligonucleotide sample (from 2 above) onto the gel. Due to diffusion of the sample during electrophoresis, it is advisable to leave an empty lane between each sample. Run gel at 30-50 watts until the bromophenol blue dye front is ~75% down the gel.

6) Separate the gel plates and transfer the gel onto Saran wrap. The oligonucleotide samples are visualized by short-wave UV shadowing; onto a TLC plate placed under the Saran wrap and gel. The area of each gel lane containing the oligonucleotide appears as a large dark spot against the green illuminated background of the TLC plate.

7) Cut out the region of the gel containing the oligonucleotide with a razor blade.

8) To elute the oligonucleotide out of the gel slices, cut each slice into ~1 cm2 pieces and place all the pieces from each slice (usually 4-6) into 5 ml of elution buffer:
Elution buffer;:
500 mM NH4OAc
1 mM EDTA
10 mM MgOAc
0.1% SDS

9) Elute oligonucleotide out of gel for 4-6 hrs at room temp (gentle agitation is optional). To check that elution is complete, examine gel pieces by short-wave UV shadowing as described above.

10) Extract the oligonucleotide eluate with phenol and ethanol precipitate. Wash the pellet 2X with 70% ethanol, dry and redissolve in a small volume (eg. 100-200 µl) of TE, ddH2O or other solution.

11) Measure the concentration of the purified oligonucleotide solution using a spectrophotometer. For oligonucleotide solutions, I assume that an OD260 of 1.0 is equivalent to 40 µg/ml.