Desalting Oligonucleotides by Gel Filtration
- Desalting or buffer exchange on a gel filtration column is dependent on volume, not quantity of DNA in solution.
- Many sizes of gel filtration columns are available. For this protocol, a NAP-10 column with a column volume of 1.0 ml is used. As long as your oligonucleotide is in solution, you won't overload the column.
- Wear gloves and eye protection when performing this procedure.
- Dissolve or bring the volume of your oligonucleotide up to 1 ml with purified water (HPLC grade or better).
Preparing Gel Filtration Column
- Using a ring stand or similar device, secure the NAP-10 column in a vertical position. Place a drip tray underneath the column to catch the waste.
- Remove the top off the NAP-10 column and empty the storage liquid into the drip tray. CAUTION: Avoid contact with the storage liquid. This solution may contain poisonous preservatives such as Kathon.
- Remove the cap off the bottom of the NAP-10 column and place the column in the stand.
- Fill the top portion of the column with purified water (HPLC grade or better). Allow all of the water to elute through the column.
- Repeat 2 more times for a total of 3 column washes.
- Pipette your oligonucleotide solution (1.0 ml volume) onto the bed of the NAP-10 column.
- Water will elute out the bottom of the column in a volume equal to your sample volume. This volume does not contain oligonucleotide and therefore does not need to be collected.
- Hold a 2 ml tube directly under the NAP-10 column to catch the sample as it elutes from the column.
- Pipette 1.5 ml purified water onto the NAP-10 column.
- Collect the the liquid that elutes out of the column in the 2 ml tube.
- When the 1.5 ml has entirely run through the column, the salt removal is complete.
- The desalted oligonucleotide is now ready to be dried or quantitated.