Enhanced Diagnostic Tools
Purification is recommended for all oligos greater than 40 bases in length and for many modified oligos. For demanding applications such as multiplex PCR, cloning, mutagenesis or antisense/RNAi methods, additional purification will significantly improve oligonucleotide performance. Your choice of purification should be based on several factors including the application you will be using the oligo in, the length of the oligo, and whether you have any modifications on the oligo. Oligos are synthesized base by base using chemical reactions which are only ~99% efficient, so with each base addition, approximately ~1% of the oligos will not pick up the proper base. We try to prevent these oligos from participating in later base additions by running through a capping reaction. Most oligos will pick up this capping reagent and will become truncation mutants. Unfortunately, because the capping reaction is not 100% efficient, some oligos will remain uncapped and able to react with later bases. This leads to the formation of deletion mutants. This can happen anywhere within the sequence and leads to a mixture of full length oligos, truncated products, and oligos with internal deletions. Part of your decision to add additional purification should be based on how sensitive your particular application is to the presence of truncated products or those with internal deletions. For some applications, such as PCR or sequencing, standard desalting is perfectly fine because the truncations and deletions will not affect your results appreciably. For other applications, such as cloning, mutagenesis, and gel shift, full length product is of utmost importance, PAGE purification should be strongly considered. PAGE will result in the highest purity level in terms of full length product, and your oligos will be at least 90% full length. PAGE purification does tend to result in lower yields than HPLC purification, however. If you need a relatively clean product, but also need a higher yield, you should consider standard HPLC for oligos up to 50nts in length, and IE-HPLC for longer oligos. IE-HPLC will result in a 90% purity level for longer oligos. You should also take the types of modifications on your oligo into account when choosing a purification method. PAGE purification can damage certain modifications, including many fluorophores and some modifications used for attachment. PAGE purification should be avoided for the following modifications: Any fluorophore, Acrydite, Amino modifiers, Biotin, Digoxigenin, I-Linker, Spacer 18, Thiol modifiers. HPLC or IE-HPLC would be the purification of choice in this case.
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