Hello,
Could you recommend a good protocol for purification of 5' biotinylated oligos that will further be used in a MS experiment (need to avoid EDTA, glycerol and other chemical contaminants)? My oligos are 125 nt long. I tried to get a quote and specific details about the effective yield (percentage of full length oligos in a, let's say, 4nmole solution), but the answers vary greatly, some companies promise as much as 38% (which is unfeasible and not likely to be true), others say 1nmole of full length in 1umole solution.
Basically, this is needed for further stoichiometric considerations.
Thank you.
Do you know a good protocol for purification of long (125 nt) biotinylated oligos for MS experiment?
Frequently, the biotinylated oligonucleotides are obtained when they are chemical synthesized by biotin phosphoramidite method (solid phase oligonucleotide synthesis. This commercial protected oligonucleotide is mostly attached to the the wall of the tube and could be detached by cleavage with Ammonium hydroxide.
Both free oligonucleotide and conjugates one could be purified using Reverse-phase HPLC or Anion-exchange HPLC. And the choices of HPLC conditions will ovoid EDTA glycerol and other chemicals.
As an example
- RP-HPLC: Elution by solvent as Water/acetonitile that could be evaporated using Speed-Vacum
- Anion Exchange HPLC,: Using gradient of Ammonium acetate buffer for elution. This salt is also evaporated in Speed vacum.
Many workers used PAGE to purify both oligonucleotide (in 15% polyacrylamides gel) and after the elution from the gel, they finish by a new purification with Oligonucleotide Sigma or Qiagen kits or Cartridges.
However, some authors use these Kits and Cartridges for direct oligo purification without use of HPLC or PAGE methods.
So you are trying to obtain only the 125mer from a mix of redundant (n-1, etc) sequences at a scale you need and without contaminants: I think thats difficult. Mohamed suggests 15% PAGE, and indeed a long (very long) gel would probably work, but then you have to locate the band and elute, and yes, repurifiy away from low molecular weight contaminants. I dont Ithink the yield would be sufficient unless you run several gels or lanes. I dont think RP-HPLC or MEC will give you high enough resolution.
Have you considered generating the oligo by PCR?
Thank you Geoff and Mohamed,
I settled for Gblocks and manual biotinylation, as commercial biotinylated oligos are pricey and may not work.
Good idea. But I would hav ethought that the commercial biotinylation is actually cost effective: you wont need any further purification and if the process isnt 100% efficient (nor would be TdT addition) it doesnt matter because the non-biotin oligos would not be captured.
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