How should oligos be purified for PCR?
Posted February 13, 2023
You can use a few different techniques for purifying oligos for PCR. The right technique will depend on the amount to be purified as well as the degree of purification required for the application. These are some of the techniques you can use for purification of oligos for PCR.
Among the most basic of oligo purifying processes, desalting involves using normal phase chromatography to remove the excess salt from your mixture. This yields a salt-free, ready to use DNA solution that is excellent for robust techniques such as PCR, microarrays, and amplification restriction fragment polymorphism (ARFP) analysis among others. Desalting can be used in combination with other purification methods.
Cartridge purification removes failure sequences from your final mixture using reverse phase chromatography. This technique results in fairly pure material, which is excellent for PCR, screening, cloning, or preliminary screening purposes.
PAGE (polyacrylamide gel electrophoresis) uses a denaturing environment to separate oligos according to molecular weight. This is the only purification method that can resolve unmodified oligos with lengths greater than 80 bases. The limitations of this technique are the relatively poor yields and the ability to purify only small amounts of oligo at a time. This technique is recommended for sequences ≥50 bases and when high purify is required.
RP HPLC (reverse phase high pressure liquid chromatography) uses hydrophobicity to remove failure sequences from the sample and isolate full-length oligonucleotides. It is considered the gold standard for purifying large amounts of oligos at high purity and is the preferred technique for a large number of downstream applications. The only limitation of RP HPLC is that it cannot resolve oligos greater than 40 bases or less than 8 bases.
AEX HPLC (Anion-exchange high pressure liquid chromatography) is based on the number of charged groups (phosphate) in the oligonucleotide backbone. It is the go-to technique for purifying smaller quantities of unmodified oligonucleotides up to 80 bases. Using this method for longer oligos results in lower and less consistent purity because of the lower resolution between the full-length sequence and shortmers.
No single oligo purification technique is best for all applications. Each one has its own strengths and weaknesses. The best technique for you will depend on three major factors: oligo length, the amount of purified oligos you need, and how pure you need it to be for your application.