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Purification

For demanding applications such as multiplex PCR, cloning, mutagenesis or antisense/RNAi methods, additional purification can significantly improve oligonucleotide performance. Purified oligos up to 60 bases that are provided with a purity guarantee receive QC by capillary electrophoresis (CE). The traces are available online in your account order history.

Purification services offered include PAGE and various types of HPLC. Purified, unmodified oligos 20–50 bases in length are provided with the guaranteed yields (Table 1).*

Table 1. Yield guarantees for unmodified oligos of 20–50 bases, after purification.

Purification type100 nmol250 nmol1 µmol5 µmol10 µmol
PAGE1 OD2 OD10 OD50 OD100 OD
HPLC1 OD
4 OD20 OD100 OD200 OD
IE-HPLC1 OD
4 OD20 ODs100 OD200 OD
RNase-Free HPLC1 OD
4 OD20 ODs100 OD200 OD
Dual HPLC1 OD
2 OD10 ODs50 OD100 OD
Dual PAGE & HPLC0.5 OD1 OD5 ODs25 OD50 OD

* Purity and yield guarantees vary with oligo length, modifications, or sequence composition.

HPLC purification

High performance liquid chromatography (HPLC), a form of column chromatography, can be performed in one of two ways. Reverse-phase (RP) HPLC separates full-length oligo product from truncated products based on relative hydrophobicity. Ion-exchange (IE) HPLC separates full-length oligonucleotides from truncated species based on relative charge difference. IDT uses HPLC to purify unmodified oligos as well as oligos with complex modifications such as linkers, spacers, modified bases, and hydrophobic modifications. The type of HPLC selected is dependent on oligo sequence composition and characteristics.

PAGE purification

Polyacrylamide gel electrophoresis (PAGE) separates full-length product from shorter species based on electric charge. PAGE purification is most effective for unmodified oligos that only need truncated product removed. It substantially reduces the amount (mass) of final oligo product; however, the dramatic increase seen in purity justifies the smaller yield. We strongly recommended PAGE purification for all oligos >60 bases in length. Purity of >90% is routinely achieved, but may vary due to oligo length, modifications, or sequence composition.

RNase-Free HPLC purification

Originally developed for isolating RNA oligos, RNase-Free HPLC purification has been extended to DNA oligos for use in applications with a high sensitivity to ribonucleases. RNase-Free HPLC purification is performed in an RNase-free environment: reagents, equipment, and lab surfaces are monitored for RNase contamination using RNaseAlert® reagent.

Dual purification

For applications that demand the utmost in oligo purity, we offer dual HPLC purification or dual PAGE and HPLC purification. These methods result in oligos of the highest possible purity. Dual HPLC purification can be used to purify dual-labeled probes and Molecular Beacons bearing NHS ester modifications.

Which purification method?

The purification method you need depends on oligo composition and your research application. If you have questions regarding which type of purification is suitable for your oligos, our experts can provide guidance. Contact us at applicationsupport@idtdna.com.