Protocol

Initial purification of DMT-oligonucleotides

Cleavage of oligodeoxynucleotides from column and deprotection.

The oligodeoxynucleotide could have been synthesized on several types of columns. We have used the standard column and the Universal II column. The Standard column comes with the 3'-base already on the support. The Universal Support II comes without a preformed bas attached. This allows us to place a modified nucleotide on the 3'-position.

Cleave oligodeoxynucleotide from standard columns

The oligodeoxynucleotide is cleaved from the column by incubating with concentrated NH4OH for 90 min.

1. Fill one 1 mL plastic syringe that has a rubber tipped piston with concentrated NH4OH.
2. Fit this syringe on one end of the column
3. Fit a second empty syringe on the other end
4. Make sure the syringes are tightly on the column
5. Hold both syringes Push the NH₄OH through the column several times. Repeat this every 10-15 min for 90 min total incubation time.
Note: NH₄OH must be 28% by weight. This is ensured by using NH₄OH stored in the freezer. NH₄OH, from a newly opened bottle, is pipetted into 1.5 mL microcentrifuge tubes and stored in the freezer.
6. Place in screw-top microcentrifuge tube and incubate at 55 C overnight.
7. Let cool to room temperature

Cleave oligodeoxynucleotide from Universal II columns

The oligodeoxynucleotide is cleaved from the column by incubating with 2 M NH₃/MeOH for 30 min.

1. Prepare concentrated NH₃ by bubbling NH₃ through MeOH for 5 min.
2. Fit an empty syringe on one end of the column
3. Fill one 1 mL plastic syringe that has a rubber tipped piston with NH₃/MeOH.
4. Fit this syringe on the other end of the column
5. Make sure the syringes are tightly on the column>
6. Hold both syringes
7. Push the NH₃/MeOH through the column several times.
8. Repeat this every 5 min for 30 min total incubation time.
   Note: NH₃ must be at least 2 M, although higher concentrations are good.
   
9. Place in screw-top microcentrifuge tube and evaporate
   
10. Add 1 mL concentrated NH4OH and incubate at 55 C overnight.
    
11. Let cool to room temperature
    

Sep pak preparation and detritylation

Prepare C-18-sep pak columns

1. Wash with 10 mL methanol to wet Sep Pak
   Sep pak must be wetted with a non-polar solvent
2. Wash with 5 mL 40%methanol/water
3. Wash with initial buffer, 10 mL 100 mM Et₃N-HOAc, pH 7.0

Crude purification of oligodeoxynucleotide

1. Evaporate NH₄OH
2. Dissolve oligodeoxynculeotide in water and load onto  Sep-pak.
   Save eluant in same centrifuge tube and reload 3X
3. Wash with 7 mL 3% NH₄OH to elute failure sequences
4. Wash with 7 mL H₂O to wash out NH₄OH
5. Wash with 5 mL 2% CF₃COOH to remove DMT group
6. Wash with 5 mL H₂O to wash out acid
7. Elute oligodeoxynucleotide with 40% MeOH/H₂O
   " Discard first 5 drops (combine with previous eluant)
   " Collect 2 mL in two different microcentrifuge tubes
   Note: The elution must be done slowly to ensure equilibrium conditions. 1 drop per second
8. Evaporate solvent with centrifugal evaporator

Crude purification with PolyPak columns

Prepare PolyPak II columns

See brochure from Glen Research

The polyPak II columns can be used for the 1 mol scale synthesis.

1. Wash with 4 mL acetonitrile to wet column
2. Wash with initial buffer, 4 mL 2 M Et₃N-HOAc, pH 7.

 
Crude purification of oligodeoxynucleotide

1. Dilute NH₄OH solution with three volumes of H₂O. Up to 10 mL may result.
2. Load oligodeoxynucleotide in NH4OH onto PolyPak.
   Save eluant in same centrifuge tube and reload 3X
3. Wash with 7 mL 1.5% NH₄OH (1:20 dilution) to elute failure sequences.
   Use 3% NH4OH (1:10 dilution) with oligos > 35-mer.
4.  Wash with 5 mL H₂O to wash out NH₄OH
5. Wash with 5 mL 2% CF₃COOH to remove DMT group
6. Wash with 5 mL H₂O to wash out acid
7. Elute oligodeoxynucleotide with 20% CH3CN/H2O
   " Discard first 3 drops (combine with previous eluant)
   " Collect 2 mL in two different microcentrifuge tubes
   Note: The elution must be done slowly to ensure equilibrium conditions. 1 drop per second
8. Evaporate solvent with centrifugal evaporator

Desalting with Sep paks

Prepare C-18 Sep Pak

1. Wash with 10 mL methanol to wet Sep Pak
2. Sep pak must be wetted with a non-polar solvent
3. Wash with 5 mL 40%methanol/water
4. Wash with initial buffer, 10 mL 100 mM Et₃N-HOAc, pH 7.0

Desalt

1. Dissolve oligonucleotide in about 1 mL 100 mM Et₃N-HOAc, pH 7.0
   interactions between phosphate and Et3N help keep oligodeoxynucleotide bound to Sep Pak
2. Load sample three times using a flow rate of about 1 drop/second
3. Wash sample with 3 mL 100 mM Et₃N-HOAc, pH 7.0
4. Elute sample with 40% methanol/water

HPLC Purification of oligodeoxynucleotides