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You are at: All > Oligonucleotides > Custom Oligonucleotide Components > Custom Oligonucleotide Components by Type > Purification Methods

Double RP-HPLC Purification

 



Double RP-HPLC Purification

The most commonly requested method for oligonucleotide purification is high performance liquid chromatography (HPLC). Reverse phase (RP) HPLC, which separates on the basis of lipophilicity to mass, is generally used because of cost and capacity. Our HPLC purifications always start with a trityl-on purification, and are often followed with a second full gradient trityl-off HPLC for an extra level of purification. For our single RP-HPLC purification, the initial trityl-on purification is entirely sufficient and a cartridge is utilized in the subsequent trityl-off oligonucleotide processing. This process can yield highly pure material, especially when the oligonucleotide is not complex.
 
However, when synthesizing compounds that require a post-synthetic modification, such as a dye label, we will perform an additional RP-HPLC purification. Unfortunately, even a rigorous RP-HPLC process like ours cannot separate all the side products formed during oligonucleotide synthesis in every case. The method is even further challenged by the complexities added from the synthesis of highly modified species. In those cases we will often initially purify the oligonucleotide using an orthogonal method, usually PAGE.
 



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Reference(s)
Jahic H, Liu CF, Thresher J, Livchak S, Wang H, Ehmann DE. The kinetic mechanism of S. pneumoniae DNA ligase and inhibition by adenosine-based antibacterial compounds. Biochem Pharmacol. 2012 Jun 25. doi: 10.1016/j.bcp.2012.06.017.
Clark TA, Spittle KE, Turner SW, Korlach J. Direct Detection and Sequencing of Damaged DNA Bases. Genome Integr. 2011 Dec 20;2(1):10.


 

 


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