Purify Oligo (Day1)

1. Resuspend oligo in 300 uL of sterile water (or use 300 uL of 100 uM oligo).

2. Add 300 uL of formamide (or 80% formamide 1X TBE).

3. Heat for at least 5 min at 90 deg C.

4. Pre-run gel for ~30 min at 300V.

5. Run on 16% SequaGel (large spacers = 1.5mm)

. 100 uL sample/lane

. run 1 lane of 2X Protein Loading Buffer

. run for ~2.5hrs at 300V.

6. Lay gel on TLC plate covered in Saran Wrap.

7. Briefly expose gel to UV light to visualize bands.

8. Cut out bands and place in 3 cc syringe.

9. Push gel slices through the syringe to break into small pieces.

10. Use 1mL Oligo Elution Buffer to wash the syringe and add 4mL Oligo Elution Buffer to the 15mL conical tube.

11. Incubate O/N at 37 deg C, shaking.

OLIGO ELUTION BUFFER:

0.5 M Ammonium Acetate

10mM Magnesium Acetate

0.1% SDS

Purify Oligo (Day2)

1. Remove as much liquid as possible.

2. Spin @ 3500 rpm for 5 min at RT.

3. Remove remaining liquid and add to the 1^st decant.

4. Pass oligo solution through 0.45 um nylon filter.

5. Wash filter with 1mL oligo elution buffer.

Prepare Sep-Pak C₁₈ Column:

6. Equilibrate with 10mL HPLC grade acetonitrile (push through slowly)

10mL sterile water and

2mL 10mM ammonium acetate

7. Push through sample and save effluent.

8. Wash with 10mL sterile water 3X and save effluent.

9. Elute oligo with 1mL 60% methanol into Eppendorf tube 3X.

MUST TURN ON SPEEDVAC 45 MINS BEFORE USE!!!!!

10. Evaporate in speedvac (heat at med., RC off) for 2-2.5hrs.

Resuspend in 50 uL sterile water.