Experiments on purification of radiolabeled oligonucleotides by Sep-Pak C18 column chromatography

Summary

This scheme describes a method to separate radiolabeled oligonucleotides from undoped radiolabel using the property of antiphase affinity of oligonucleotides for silica gel. The method in this scheme can only be used to purify radiolabeled or unlabeled oligonucleotides containing phosphate groups at the 5' end, whereas the method described in Scheme 1 is mostly used for oligonucleotides with free hydroxyl groups at the 5' end. This experiment is from Molecular Cloning Laboratory Guide (3rd Edition), Book 1, by Peitang Huang.

Operation method

Experiments on purification of radiolabeled oligonucleotides by Sep-Pak C18 column chromatography

Materials and Instruments

Acetonitrile Ammonium bicarbonate TE solution Radiolabeled oligonucleotides
Centrifuge dryer Microcentrifuge tubes in racks or traps Sep-Pak classic chromatography columns Syringes

Move

makings

Solutions and buffers
Dilute the storage solution to the appropriate concentration.

Acetonitrile (5%, 30% and 100%)
For each Sep-Pak C18 column, use 10 ml of HPLC grade acetonitrile (100%) and dilute the acetonitrile with water before use.

Ammonium bicarbonate (25 mmol/L, pH 8.0)

Ammonium bicarbonate (25 mmol/L, pH 8.0) with 5% (WV) acetonitrile.
Mix 5 ml of acetonitrile with 95 ml of 25 mmol/L ammonium bicarbonate.

TE solution (pH 7.6)

Nucleic acids and oligonucleotides

Radiolabeled Oligonucleotides

The raw material used for purification is the reaction mix of Scheme 2 (step 3 or step 5) and has been heat inactivated at 68°C for phage T4 polynucleotides.

Specialized equipment

Centrifugal dryer (Savant Speed Vac or equivalent)

Microcentrifuge tubes (1.5 ml) in racks or traps
For collection of radiolabeled oligonucleotide eluates for chromatography column purification

Sep-Pak Classic Chromatography Columns, Short Bodies
Each Sep-Pak Classic column (available from Waters, Millipore) contains 360 mg of hydrophobic reversed-phase chromatography resin (C18). Purification utilizes the principle that oligonucleotides bind to the resin when the solvent (aqueous solution) is more polar and detach when the melt (e.g., methanol-water mixture) is less polar. A separation column is required for each phosphorylation reaction.

Syringe (10 ml polypropylene syringe)

Methods

1. Sep-Pak Cl8 reversed-phase chromatography columns are prepared as follows:

a. Attach a polypropylene syringe containing 10 ml of acetonitrile to the Sep-PakC18 Classic Chromatography column.

b. Slowly rinse the column with the syringe.

c. Separate the syringe from the column, then withdraw the syringe cartridge from the syringe needle, which prevents air from being drawn into the column. Attach the syringe needle again to the chromatography column.

d. Rinse off the organic solvent with 10 ml of filtered sterile water 2 times, repeating step c after each rinse.

2. Dilute the radiolabeled oligonucleotide to 1.5 ml with sterile water and push the entire sample into the column with a syringe.

3. Rinse the column with the following four solutions and repeat step 1c after each rinse.

10 ml 25 mmol/L ammonium bicarbonate (pH 8.0)
10 ml 25 mmol/L ammonium oxygencarbonate/5% acetonitrile
10 ml 5% acetonitrile
10 ml 5% acetonitrile

4.3 Elute radiolabeled oligonucleotides in lml of 30% acetonitrile and collect each fraction separately in a 1.5 ml microcentrifuge tube, repeating step lc after each elution.
WARNING: Phosphorylation reactions often use radiolabeled ATPs greater than 100uCi, so the dose of radioactivity from these chromatographic fractions can be significant. Unadulterated radioactive material, pipette tips, and microcentrifuge tubes should be handled with care and caution.

5. Dry the oligonucleotide-containing solution using a centrifugal dryer (Savant SpeedVac or equivalent).

6. Dissolve the radiolabeled oligonucleotide in a small volume (10 ul) of TE (pH 7.6).

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/