Oligo purification

You can choose between three options for purifying your oligo: HPLC purification, PAGE purification and Reverse-Phase Cartridge purification.

Why purify?

In spite of the highly efficient coupling efficiency achieved by Biolegio (a typical synthesis step yield of 99,5%), around 0,5% of all extension fails. This leads to truncated synthesis products with the longest of them being (n-1) mer. The relative proportion of undesired material increases as oligo length increases. For some applications it will be desirable to enrich the final product in full length oligonucleotides.

Which kind should I choose?

Which kind of purification is chosen largely depends on your demands concerning purity and yield of the end product. You must consider that purification results in less yield of the final product. PAGE purification, although resulting in a higher purity, generally gives lower yields than the HPLC purification. The kind of purification which is chosen depends also on the length of an oligo. For oligos with a length over 50 bases we recommend PAGE purification to guarantee purity of the final product.

Reverse-Phase cartridge purification

Purification with Reverse-Phase cartridge offers the lowest level of purity (typically 80%). The basis of the separation is the difference in hydrophobicity between full length product (which contains a 5’-DMT) and truncated sequences (without DMT groups). Because the differences in hydrophobicity between the full length DMT product and non-DMT truncated sequences are reduced as the oligo length is increased, cartridge purification is not recommended for oligos > 50 bases.

HPLC Reverse-phase purification

Reverse-phase HPLC operates on the same principle as the reverse-phase cartridges, but typical yields a product of 90% purity. The capacity and resolving properties of HPLC columns are also much greater than cartridge devices. Therefore, HPLC is the method of choice for purifying larger quantities of oligos (i.e. >= 1 μmol). As with cartridges, reverse-phase HPLC is usually not recommended for purifying oligos longer than 50 bases.

PAGE Purification

With the excellent resolution of PAGE a 95-99% purity can be achieved. The basis of the separation is charge and molecular weight. In most cases, a full length (n) oligo can be separated from oligos only one base shorter (n-1). PAGE is the recommended technique for purifying oligos over 50 bases long. Yields from PAGE are lower than from other methods due to the relative inefficient extraction of oligos from the gel.