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20 22 6

15 30 9-10

12 40 approx. 15

11. Lay the gel mold flat on plastic-backed protective bench paper with the smaller (notched) plate uppermost. Allow the gel to cool to <37°C before proceeding.

12. Remove any remaining pieces of electrical tape. Use a spacer or a plate-separating tool to slowly and gently pry apart the plates of the mold. The gel should remain attached to the longer (nonsiliconized) glass plate.

If the gel adheres to both plates, replace the partially dislodged, smaller or notched plate back on the gel, invert the plates, and try again.

13. Place a piece of Saran Wrap on the gel, turn the glass plate over, and transfer the gel to the Saran Wrap. Place a piece of Parafilm or a fluorescent thin-layer chromatographic plate under the gel where the oligonucleotide is predicted to be.

14. Use a hand-held UV lamp to examine the gel by illumination from above at 260 nm.

The DNA in the gel absorbs the UV radiation and appears as dark blue bands against a uniform fluorescent background contributed by the Parafilm or chromatographic plate. If the DNA is difficult to visualize, take the gel into a darkened room and illuminate it with the hand-held UV lamp.

15. Recover the desired oligonucleotide, which should be the slowest-migrating band (i.e., closest to the top of the gel), by excising each DNA band with a sharp, clean scalpel or razor blade. Avoid taking UV-absorbing material smaller in length than the desired oligonucleotide.

16. Transfer the gel slices to three or four microfuge tubes. Add 1 ml of oligonucleotide elution buffer to each tube. Crush the slices with a disposable pipette tip, using a circular motion and pressing the fragments of gel against the sides of the tubes. Seal the tubes well. Incubate the tubes for 12 hours at 37°C in a shaker incubator.

17. Centrifuge the tubes at maximum speed for 5 minutes at room temperature in a microfuge. Pool the supernatants, transfer them to a 5-cc disposable syringe, and pass them through a Millex HV filter. Collect the effluent in a 15-ml polypropylene tube.

18. Prepare a Sep-Pak C13 reversed-phase column as follows:

a. Attach the barrel of a disposable 10-cc polypropylene syringe to the longer end of a Sep-Pak C18 classic column.

b. Add 10 ml of acetonitrile to the barrel and slowly push it through the column with the plunger of the syringe.

c. Remove the syringe from the Sep-Pak column and then take the plunger out of the barrel. This prevents air being pulled back into the column. Reattach the barrel to the column.

d. Add 10 ml of sterile filtered H2O (Milli-Q or equivalent) to the barrel and slowly push it through the column with the plunger. Repeat Step c.

e. Add 2 ml of 10 mM ammonium acetate to the barrel and push it slowly through the column. Again remove the syringe, remove the barrel, and reattach the barrel to the column. The column is now ready for use.

19. Add the solution containing the gel-purified oligonucleotide (from Step 17) to the barrel and slowly push it through the column with the plunger. Collect the effluent in a sterile 50-ml polypropylene tube. Repeat Step 18c.

20. Add 10 ml of H2O to the barrel and push it slowly through the column with the plunger. Repeat this wash step twice more.

21. Elute the bound oligonucleotide from the Sep-Pak column with three aliquots of 1 ml of methanol:H2O solution. Repeat Step 18c after each elution. Collect each effluent in a separate microfuge tube. Read the OD260 of the solution in each of the three microfuge tubes, using the methanol:H2O solution as a blank. More than 90% of the oligonucleotide applied to the column should elute in the first fraction.

22. Evaporate the solution containing the oligonucleotide to dryness in a centrifugal evaporator.

23. Dissolve the oligonucleotide in a total volume of 200 pl of H2O or TE (pH 8.0).

24. Transfer 5 pl of the solution to a cuvette containing 995 pl of H2O. Mix the contents of the cuvette, and read the OD260 of the diluted sample. Calculate the amount of oligonucleotide present in the total solution (Step 23) as described in Step 6 of this protocol.

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