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 25–50 bases, after purification.
|Purification type||100 nmol||250 nmol||1 µmol||5 µmol||10 µmol|
|PAGE||2 nmol||6.5 nmol||25 nmol||125 nmol||250 nmol|
|HPLC||3.75 nmol||12.5 nmol||50 nmol||250 nmol||500 nmol|
|IE-HPLC||3.75 nmol||12.5 nmol||50 nmol||250 nmol||500 nmol|
|RNase-Free HPLC||3.75 nmol||12.5 nmol||50 nmol||250 nmol||500 nmol|
|Dual HPLC||2 nmol||6.5 nmol||25 nmol||125 nmol||250 nmol|
|Dual PAGE & HPLC||0.5 nmol||3 nmol||12.5 nmol||62.5 nmol||125 nmol|
* Purity and yield guarantees vary with oligo length, modifications, or sequence composition.
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.
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.
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.
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.