Purification Options

There are several options available for purification of oligonucleotides. The NACF offers two forms of purification. Any oligo can be desalted, and oligos which are 50mers or shorter can be purified utilizing trityl-on synthesis and cartridge purification. For oligos longer than 50 nucleotides we suggest either PAGE or HPLC purification. The two types of purification that we perform are described below.

1) Trityl-On Purification:

During oligo synthesis, each coupling step has an efficiency of 98 to 99%. The 1-2% of oligos that do not couple at each step are inactivated and remain in the final synthesis product; these are called n-minus products. In long oligos, the amount of n- material can be significant. As an example, consider a 75-mer synthesized with 99% efficiency at each step. The amount of full length product will be (.99)75 = 47%. The remaining 53% of the final synthesised product consists of n- products. Because synthesis proceeds from 3' to 5', the n- products consist of deletions from the 5' end. For many applications, this large amount of n- product is not a problem. Occasionally, however, the n- product will interfere and must be removed. Before synthesis starts the NACF can set the machines to add the trityl groups. Purification can then be accomplished by leaving the trityl group on the 5' nucleotide at the end of synthesis. The details of the trityl group would take several paragraphs to explain; suffice it to say that if the "Trityl On" option is selected, only full length synthesis products will contain a trityl group. Since only the full-length oligo will have a trityl group, it can be purified away from n- products through the use of a small chromatography cartridge under conditions that favor trityl binding. Non-trityl containing oligos (the n- products) are not bound and are washed away. The trityl group is then chemically cleaved and remains bound to the cartridge while the full-length oligo is eluted and collected. Contaminating salts are also removed during this process.

Please note that the yield of a purified oligonucleotide will be considerably lower than that of a crude oligo. Typically 1 to 4 OD units will be obtained for purified oligos.

Click here for Trityl-On purification protocol.

2) SepPak Desalting

In order to Desalt an oligo, the DNA is loaded onto a cartridge in an aqueous solution and binds to the matrix of the cartridge while contaminating salts and very small failure sequences are washed through the cartridge. The oligo is then eluted using a methanol/water soution then dried for storage.

Click here for SepPak desalt protocol.