We offer several different purification schemes. They offer a range of capabilities and applications. Our most common method is reverse phase (RP) HPLC. We use C-18 columns for most of our work. The vast majority of our compounds are at least double purified, first undergoing a trityl-on prep, followed by another full HPLC purification after removal of the trityl group. This ensures a high level of both length and chemical purity, which is comparable or even superior in many cases to more sophisticated and costly anion exchange (AX) HPLC methods. We have demonstrated this to many customers and would be happy to demonstrate the same to you with a sample synthesis. We do offer AX-HPLC, but in a more limited capacity. Another popular method is polyacrylamide gel electrophoresis (PAGE). This method is limited to just a few tens of milligrams of material (15 μmole starting scale) and becomes expensive very quickly. It is a good method to enhance length purity, but is limited in its ability to remove chemical contaminants.
It is very difficult to remove all excess salt. However, we do routinely obtain less than 5% excess salt, and our diafiltration process for larger samples can reduce the salt content to even lower amounts.
Dyes listed with the “extra purification step” requirement are made in a two-step process. First, the unlabeled oligonucleotide with the appropriate amino or thiol linker is synthesized and then purified to isolate only the full-length oligonucleotide. The oligonucleotide is then conjugated to the dye and purified again to remove any unlabeled material. Some fluorescent dyes, such as 6-FAM, TET and HEX, do not require this extra purification step. These dyes are available as phosphoramidite reagents, allowing us to label the oligonucleotide during the synthesis procedure and use a single purification step.
The need for oligo purification is dependent on a number of factors, including your application and oligo complexity. HPLC and PAGE are the most common purification methods. Reverse phase HPLC (RP-HPLC) is by far the most common, and is very useful in separating dye-labeled from unlabeled oligos, and in separating full-length oligos from truncated species (n-1, n-2, etc.). A crude/desalted preparation of a primer is usually sufficient for a PCR application. Long oligos (45+ bp) do not resolve well by HPLC, therefore PAGE purification is recommended. RNA requires purification under sterile conditions and sterility is most easily controlled during a PAGE purification procedure. Our technical support team can assist you in determining the best purification method for your construct and requirements.