Protocol

Desalting with Sep paks

Prepare C-18 Sep Pak

1. Wash with 10 mL methanol (or acetonitrile) to wet Sep Pak
   Sep pak must be wetted with a non-polar solvent
2. Wash with 5 mL 40%methanol/water (or 30% acetonitrile/water)
3. 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
   Collect eluant in fraction 1
3. Wash sample with 5 mL water (1 drop/sec)
   Collect this eluant in fraction 2
4. Elute sample with 40% methanol/water (or 30% acetonitrile/water) at 1 drop/sec
   • Collect first 4 drops in fraction 2
   • Collect next 1 mL in fraction 3 and 2nd mL in fraction 4.
5. Measure UV absorption at 260 nm for all four fractions