When ordering their oligo from Retrogen, many people wonder how pure is pure enough. Our high coupling efficiency proprietary synthesis platforms together with our rigorous quality control procedure result in high quality oligo that can be use in most basic molecular applications without additional purification. This technical information will help you select the best purification option for your oligo and application.
In the DNA synthesis, each nucleotide is coupled sequentially to the growing chain. In each couple cycle, a small percentage of the oligo chains will not be extended, resulting the mixture of full length and truncated oligo. After the oligo is cleaved from the support and the protecting groups are removed, there are different types of purification can be used to separate the truncated oligo from full-length oligo. For some applications, it is crucial that only full-length oligo is present in the final yield. For others, the presence of truncated oligo will not affect the experimental results.
Desalting Purification
At Retrogen, every oligo is desalted by Sephadex column. Desalting procedure removes residual by-products from the synthesis, cleavage, and deprotecting procedures. This level of purify is adequate for most common applications, such as standard PCR, sequencing, and hybridization.
Reverse-phase cartridge purification (RP1)
Separation on a reverse-phase cartridge purification offers the next level of typically 90-95 % purity. The level of purity for RP1 purification is almost equivalent to that provided by HPLC, but the recovery is much higher. The principle of this purification is based on the selection of full length product with the DMT group. The n-1 oligo without the DMT group will not be selected when the oligo is passed through the column. This purification is recommended for purifying the oligo less than 50 bases. HPLC Purification
HPLC is recommended for the oligo from 6-50 bases in length that requires additional purification. HPLC purification removes most truncated oligo sequences, resulting mostly full-length product in the final yield. The ion exchange HPLC is used to separate the oligo sequences on the basis of charge and it is performed on the Oigo3 column from Perseptive Biosystems company. From the chromatogram, the fractions with the highest purity are selects. The HPLC purified oligo is then desalted by Sephadex column.
PAGE Purification
PAGE is recommended for the oligo longer than 50 bases that required additional purification. PAGE purification removes most the truncated oligo sequences resulting the highest level of 95-99% purity, but gives lower yield. PAGE separates the oligos on the basis of charge and molecular weight. PAGE purification is performed on a denaturing polyacrylamide gel prepared from stock solution of acrylamide, urea, and TBE buffer. The oligo is detected through UV shadowing onto the sensitive digital image system. The full-length product is excised and eluted from the gel. The PAGE purified oligo is then desalted by Sephadex column.
Recommended Scale of Synthesis and Purification Levels
1-800-RETROGEN
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