FAQ 5: Do the oligos I order need to be purified?

ANSWER 5: Not necessarily, it depends on what they are being used for. We order our oligos from IDT as “salt free” but otherwise unpurified. However, the manufacturers do suggest further purification for long oligos, such as the 70bp ones we use for oligo-mediated recombination. The reason is that during the synthesis of oligos, the coupling reaction is about 99% efficient and thus 1% of oligos do not pick up the proper base. After each step, attempts are made to keep these “mutant” oligos from extending by use of a capping reagent. However, this process is not 100% so some oligos that are missing a given base continue on to the next addition step creating a deletion-containing oligo.  From IDT tech support, a 70mer is 55% full length and 45% truncated/mutated products. That said, in experiments it was shown that about 2% of 82mers contained mutations after recombineering (see Oppenheim reference below*). Further, we normally have a selection for recombinants (e.g. Gal+) so the selection process itself “weeds out” any oligos containing mutations that don’t allow the cells to become Gal+.  If you do not have a selection for the change you are making or the screen is difficult, purification may be worth the added expense. For oligos longer than 50bp, purification should be by PAGE to reach the highest purity as HPLC has been shown to not help much.  PAGE can increase purity to 85-90%.


*Oppenheim, A. B., Rattray, A. J., Bubunenko, M., Thomason, L. C. & Court, D. L. (2004). In vivo recombineering of bacteriophage λ by PCR fragments and single-strand oligonucleotides. Virology 319, 185-189.


 © RECOMBINEERING 2016