Technical Brief - Purification of 6-FAM Labelled oligos using Glen-Pak™ Cartridges

The Glen-Pak™ purification cartridges introduced at the end of 2007 have been very well received and we continue to add product specific protocols for use with these versatile tools for oligonucleotide (DNA and RNA) purification.

The most powerful feature of these columns is their high affinity for dimethoxytrityl (DMT)-On oligonucleotides along with their concurrent inefficient binding of DMT-Off fragments. Recently work was done with 6-Fluorescein Phosphoramidite (10-1964-xx), which conveniently has a DMT group that can be utilized for successful purification on the Glen-Pak DNA cartridge.

A mixed base 18 mer oligonucleotide was synthesized at the 1.0µmole scale and the 6-Fluorescein was added at the 5' end (DMT-On) using standard methods as described in its product profile (http://www.glenresearch.com/ProductFiles/10-1964.html).

The oligonucleotide was cleaved in 1.0mL NH4OH and deprotected overnight at 55°C. After cooling, a small portion of the crude sample was detritylated and analyzed using ion-exchange HPLC to illustrate the base protecting groups and the number of failure sequences present (Figure 1).

The balance of the deprotected oligo was diluted with an equal volume of 100mg/mL NaCl and loaded on a properly prepared Glen-Pak DNA purification cartridge (60-5200-01). After rinsing with 2 mL of salt wash solution, 2 mL of 2% TFA, and 3 mL of deionized water, the purified oligo was eluted in 1 mL of 50% Acetonitrile /Water containing 0.5% NH4OH. The eluent was dried in a speedvac, reconstituted in 0.5% NH4OH in deionized water and analyzed by ion-exchange HPLC.

Figure 2 details the dramatic enhancement in purity of the oligo post Glen-Pak purification. The crude sample contained only 77% full-length product while the final product measured 99% pure with virtually quantitative recovery (data not shown).

We plan to continue developing new protocols and confirming the use of various modifiers/labels with the Glen-Pak DNA and RNA cartridges. Feel free to contact the technical support team for advice or to give feedback about this exciting addition to our purification product line.

Glen-Pak´┐Ż is a trademark of Glen Research Corporation.

Figure 1: IEX HPLC ANALYSIS OF A CRUDE, DETRITYLATED 18 MER

fig1

Figure 2: IEX HPLC ANALYSIS OF SAME 6-FAM-LABELLED 18 MER GLEN-PAK PURified

fig2

IEX HPLC Conditions
Column: Dionex DNAPac PA200 250 x 4mm
Buffers: A - 25mM Tris, pH 8, 5% ACN;
B - 25mM Tris, pH 8, 5% ACN,
1.0M NH4Cl, pH 8
Gradient: 0-90% Buffer B at a flow rate of 1mL/min.

 

A booklet, Glen-Pak™ Cartridges, DNA and RNA Purification, describes in detail the process and several applications.

 

Glen-Pak™ is a trademark of Glen Research Corporation.

Figure 3: Structure of 6-Fluorescein Amidite

10-1964

ORDERING INFORMATION