OLIGO CLEAN UP

DEPROTECTION

1) Screw column onto a tube of Deprotection solution (NH4OH), invert and pump the syringe several times, then draw the solution onto the column.

2) Leave the column and syringe inverted for 5 min., then push the solution into the tube. Pump the syringe several times to remove all of the solution.

3) Remove and discard the column and syringe, place the tube in heating block at 65C for 5min. Chill on ice for 10 mins before opening tube.

4) Dry the oligo in the speed-vac - use heat unless drying O/N.

BUTANOL EXTRACTION

1) Resuspend the dried oligo in ~400 Ál TE.

2) Spin for 10min, transfer supernatant to a new tube.

3) Add equal volume of N-Butanol, vortex, spin ~10sec to separate the phases.

4) Remove the TOP organic phase. Some of the water will partition into the organic phase such that the aqueous phase becomes smaller with each extraction.

5) Continue to extract with EQUAL volumes of N-Butanol until ~50Ál of aqueous phase remains.

6) Dry the oligo in the speed-vac - use heat to speed this up.

MEASURE THE O.D.

1) Resuspend the dry oligo in ~200Ál TE.

2) Dilute the oligo 1:30 in TE - need a final volume of 300Ál for the cuvette.

3) Measure the O.D. at 260nm and calculate concentration as follows:

(O.D.260)x (dilution factor=30) x 33 x1000 = pmol/Ál

(330) x (length)

NB: Other labs may tell you they do not purify their oligos. They will not have accurate OD's and one day this comes back to haunt you. If you are doing really important things you should gel-purify your oligos. I do this if I am doing complex RT-PCR with degenerate oligos where the degree of degeneracy makes assessment of the [conc] important. Gel purify if it is important not to get the N-1 band.