Custom Oligonucleotide Purification

Gel Purification

All Gene Link oligos shorter than 40 mer do not require further purification if the applicaton is for PCR or sequencing. 20 mer oligo synthesized at a coupling efficiency of 99.5% will contain ~90% full length 20 mer and a mixture of truncated sequences of ~10%. As the length of the oligo increases, even at a coupling efficiency of 99.5%, the yield of the full length oligo is reducing. A 60 mer crude product will contain ~75% full-length oligo and similarly a 100 mer will contain ~60%.
Purification is strongly recommended for oligos longer than 50 mer.

Purification is strongly recommended for oligos longer than 50 mer.

HPLC/RPC

HPLC and RPC (Reverse Phase Cartridge) purification methods yield purity of 85% to 95% depending upon the sequence, GC content and length of the oligonucletide. Reverse phase based HPLC fails above 40 mer as longer oligos are inherently hydrophobic and bind non-specifically.

Polyacrylamide Gel Purification (PAGE)

Purification by this method is considered the Gold Standard for oligo purification and yields 99%+ purity. Gel purification can be used for any length of oligonucletide (as compared to HPLC and RPC cartridges which are limited to oligonucleotides below 40 mer). Gel purification is strongly advised for all applications involving cloning of the product, such as mutagenesis and gene construction applications.

Comparison of Unpurified, RPC and Gel Purified Oligos

Polyacrylamide gel electrophoresis of crude, reverse phase cartridge (RPC) and gel purified oligos. Approximately 15 ug of crude unpurified oligo were loaded to show the truncated failure sequences. Approximately 8 ug of purified oligo were loaded. Lanes 1 to 3: 68 mer; lanes 4 to 6: 56 mer. Lanes 1 & 4: crude unpurified; lanes 2 & 5: RPC purified; lanes 3 & 6: gel purified.
Results: The above gel picture shows the lack of purification efficiency of RPC as compared to gel purification. Notice the remaining truncated oligo sequences that the RPC method failed to purify.

Comparison of Unpurified and Gel Purified Oligos

Polyacrylamide gel electrophoresis of crude and gel purified oligos in adjacent lanes. Lanes 1 & 2: 63 mer; lanes 3 & 4: 96 mer; lanes 5 & 6: 175 mer; lanes 7 & 8: 43 mer.
Results: At Gene Link we recommend gel purification of all long oligos and oligos used in cloning applications. Gel purification is the gold standard method of purification as the denaturing polyacrylamide gel resolution approaches single base and the major band is clearly visible to be excised and purified.

Scale of Synthesis & Yield

Click here for RNA Oligos scale of synthesis and yield.

Post Synthesis Modifications Conjugation/Conversion (NHS, Thiol, Maleimide etc) Scale of Synthesis & Yield

Most oligo modifications are directly coupled using optimized automated DNA synthesis protocols that are suited for specific modifications. The final yield varies and are usually around 80% of the yield of the unmodified oligos. Multiple modifications and sites in the same oligo may lead to further reduction in final yield.
Certain modification (e.g Alexa fluorescent dyes, digoxigenin, etc) are not available for direct autoamted coupling chemsitry and have to be conjugated using post synthesis methods involving amino group to NHS (N - hydroxysuccinimide) ester, thiols to maleimide and other post synthesis conversions.
These post synthesis conjugation reaction kinetics yields ~60-85% final product and polyacrylamide gel purification is recommended. The yields guideline are listed below for these post synthesis conjugations.