Oligonucleotide purification using TOP-DNA, TOP-RNA and TOP-DNA Jnr cartridges

Applicable Products

Solution Preparation details

100mg/mL NaCl

• Weigh 10g of sodium chloride into a 100mL volumetric flask.
• Add water to give a total volume of 100mL.
• Mix thoroughly.

2% TFA solution

NOTE this solution needs to be made fresh on the day of use.

• Measure 2mL of TFA and add to a 100mL bottle.
• Add 98mL of water.

MeCN/water

• Measure 500mL of MeCN and add to a 1L bottle.
• Measure 500mL of water and add to the MeCN.
• Mix thoroughly.

2M Tris HCl(aq)

• Weigh out 31.53g into a 100mL volumetric flask.
• Add water to give a final volume of 100mL.
• Mix thoroughly.

1M ammonium bicarbonate / 30% MeCN

• Weigh out 7.91g into a 100mL volumetric flask.
• Add water to give a final volume of 100mL.
• Mix thoroughly.

TOP-DNA Purification

Do not remove the deprotection solution or desalt the oligo. The deprotection solution needs to be present.

1. Add 1mL of aqueous sodium chloride solution (100mg/mL) to each oligo.
2. Place one column for each oligo onto the vacuum manifold.
3. Turn the vacuum pump on.
4. Adjust the pressure to 7.0in (178mm) Hg using the vacuum control valve.
5. Add 0.5mL of acetonitrile to the cartridge.
6. Immediately then add 1mL of 2MTEAA to condition the medium.
7. Add the oligo solution to the cartridge in 1mL aliquots.
8. Add 1mL of NaCl(aq) solution (100mg/mL) to the column.
9. Add 1mL of NaCl(aq) solution (100mg/mL) to the column.
10. Add 1mL of 2% TFA/water solution to the column.
11. Add 1mL of 2% TFA/water solution to the column.
12. Add 1mL of water to the column.
13. Add 1mL of water to the column.
14. Release the vacuum.
15. Place an appropriately labelled tube beneath each of the column.
16. Add 1mL of MeCN/water 1:1 to elute the DNA oligo under vacuum.

TOP-DNA Jnr (Bond Elut®) Purification

Do not remove the deprotection solution or desalt the oligo. The deprotection solution needs to be present.

1. Add 1mL of aqueous sodium chloride solution (100mg/mL) to each oligo.
2. Appropriately label one cartridge for each oligo to be purified.
3. Pass 0.5mL of acetonitrile slowly through the cartridge.
4. Immediately then pass 1mL of 2MTEAA to condition the medium.
5. Add the oligo solution to the cartridge in 1mL aliquots by slowly passing through with a syringe.
6. Slowly pass 1mL of NaCl(aq) solution (100mg/mL) through the cartridge.
7. Slowly pass 1mL of NaCl(aq) solution (100mg/mL) through the cartridge.
8. Slowly pass 1mL of 2% TFA/water solution through the cartridge.
9. Slowly pass 1mL of 2% TFA/water solution through the cartridge.
10. Slowly pass 1mL of water through the cartridge.
11. Slowly pass 1mL of water through the cartridge.
12. Place an appropriately labelled tube beneath each of the cartridges.
13. Slowly pass 1mL of MeCN/water 1:1 to elute the DNA oligo collecting into the labelled tube.

TOP-RNA Purification (with TBDMS RNA Chemistry)

Do not remove the deprotection solution or desalt the oligo. The deprotection solution needs to be present. Use RNase free solutions for this procedure.

1. Add 1.75mL of aqueous 2M TrisHCl solution to each oligo.
2. Place one column for each oligo onto the vacuum manifold.
3. Turn the vacuum pump on.
4. Adjust the pressure to 7.0in (178mm) Hg using the vacuum control valve.
5. Add 0.5mL of acetonitrile to the cartridge.
6. Immediately then add 1mL of 2M TEAA to condition the medium.
7. Add the oligo solution to the cartridge in 1mL aliquots.
8. Add 1mL of MeCN/2MTEAA, pH7.0 solution (1:9, v:v) to the column.
9. Add 1mL of water to the column.
10. Add 1mL of 2% TFA/water solution to the column.
11. Add 1mL of 2% TFA/water solution to the column.
12. Add 1mL of water to the column.
13. Add 1mL of water to the column.
14. Release the vacuum.
15. Place an appropriately labelled tube beneath each of the column.
16. Add 1mL of 1M ammonium bicarbonate(aq)/30% MeCN solution to elute the RNA oligo under vacuum.