Desalting Oligonucleotides by Gel Filtration

General

• 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.

Sample Preparation

• 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.

Loading Oligonucleotide

• 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.

Sample Elution

• 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.