Literature (Product Manuals, Specifications, MSDS)

Catalog No.	Product	PDF Guide
R26-6400-PU	Oligo Purification

Oligo purification Gene Link provides polyacrylamide gel electrophoresis purification (PAGE) and Reverse Phase Cartridge (RPC) (as a substitute for Reverse Phase HPLC (RP-HPLC). Gel purification routinely yields 99% pure full-length product and RPC and RP-HPLC purification yields a purity of 85%-96% depending on the oligo length, base composition and sequence. The efficacy of RPC purification diminishes as the sequence length increases because retention time of a given oligo is directly proportional to its length. Higher percentage GC has the same effect due to the secondary structures that they produce. Gel purification is the gold standard of oligo purification methods and routinely yields ~99% full length product and is not dependent on size, base composition or sequence. RPC & RP-HPLC HPLC, RP-HPLC and RPC all employ a similar underlying principle, which was first used in Ion Exchange Chromatography. In this procedure, different analytes in a sample are separated based on Coulombic interactions with a stationary phase. The stationary phase is chosen to have a charge opposite that of the analytes to be analyzed. The sample containing the various analytes is then forced through the column containing the stationary phase, and they pass through with a unique retention time based on their chemistry. Instead of using the simple positive and negative charge separation technique, HPLC, RP-HPLC and RPC use polar versus non-polar separation; thus termed “Reverse Phase”. Normal Phase HPLC uses a polar stationary phase and a non-polar mobile phase, whereas RP-HPLC uses a non-polar stationary phase and a polar mobile phase. RPC purification employs the same principle as that of Reverse Phase High Pressure Liquid chromatography (RP-HPLC). Because only the full-length oligos will have the large non-polar DMT group, their retention time in the RPC column is longer than the truncated variants. This allows for the fast elution of the solvent as well as the vast majority of truncated oligos without the non-polar DMT group, while the full-length Oligos with the DMT group are retained on the column via hydrophobic interaction and then eluted separately. The difference between RPC and RP-HPLC is in the pressure applied to the column. RPC provides less pressure as it is done manually, whereas RP-HPLC is done by machine. Both methods provide nearly identical purification and are effective up to a length of 40mer. For oligos over 40mer or shorter oligos to be used in cloning or other precision downstream applications, we recommend gel purification. Gel purification separates exclusively by charge, which is equivalent to size in linearized DNA. The desired band containing full-length product is visualized, and then physically removed and dissolved so there is no chance of impurities. Gel Purification