Custom Oligonucleotide 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.
| Product | 50 nmol scale | 200 nmol scale | 1 umol scale | 2 umol scale | 10 umol scale | 15 umol scale |
| Maximum Size | 49 mer | 99 mer | 180 mer | 250 mer | 250 mer | 250 mer |
| Purification Grade | PAGE or RPC up to 35mer | PAGE | PAGE | PAGE | PAGE | PAGE |
| All Gene Link oligos shorter than 40 mer usually do not require any further purification if the application is for PCR or sequencing. | ||||||
| Product | 50 nmol scale | 200 nmol scale | 1 umol scale | 2 umol scale | 10 umol scale | 15 umol scale |
| Catalog No. (PAGE) | 26-6400-77 | 26-6400-77 | 26-6400-55 | 26-6400-88 | 26-6400-44 | 26-6400-99 |
| Gel Purification (PAGE) | 75.00 | 75.00 | 150.00 | 280.00 | 1500.00 | 1800.00 |
| Catalog No. (RPC) | 26-6400-11 | 26-6400-11 | 26-6400-22 | 26-6400-23 | 26-6400-33 | 26-6400-34 | Reverse Phase Purification (RPC) | 30.00 | 30.00 | 90.00 | 170.00 | 750.00 | 900.00 |
Certain experiments cannot wait for an extra day. When such is the case, order "Rush Oligo Purification Service". The custom oligo will be on fast track to be gel purified as soon as the oligos are synthesized and processed. This typically will save 2-3 days.
| Product | 50 nmol scale | 200 nmol scale | 1 umol scale | 2 umol scale | 10 umol scale | 15 umol scale |
| Catalog No. (PAGE) | 26-6400-77R | 26-6400-77R | 26-6400-55R | 26-6400-88R | 26-6400-44R | 26-6400-99R |
| Rush PAGE Service Charge | 35.00 | 35.00 | 50.00 | 65.00 | 250.00 | 350.00 |
| Catalog No. (RPC) | 26-6400-11R | 26-6400-11R | 26-6400-22R | 26-6400-23R | 26-6400-33R | 26-6400-34R | Rush Reverse Phase Purification (RPC) Service Charge | 10.00 | 10.00 | 15.00 | 20.00 | 40.00 | 60.00 |
Click here for RNA Oligos scale of synthesis and yield.
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.
| Length | Page | HPLC/RPC |
| 8-40 mer | Yes | Yes |
| 41-250 mer | Yes | No |
| All Gene Link oligos shorter than 40 mer usually do not require any further purification if the application is for PCR or sequencing. | ||
| Application | Purification |
| PCR and Sequencing | Not Required |
| Cloning and Gene Construction | Yes |
| Mutagenesis | Yes |
| Modified Oligos | Yes |
| Probes | Yes |
| Crude Desalted | RPC Purified** | Gel Purified | |||||||||||||
| 20 mer oligo Typical Yield |
30 mer oligo Typical Yield |
50 mer oligo Typical Yield |
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Scale |
A260 Units |
nmols |
mg |
A260 Units |
nmols |
mg |
A260 Units |
nmols |
mg |
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50 nmol |
8-10 | 30+ | 0.2-0.3 | 4-5 | 12+ | 0.1-0.16 | NR* [1-2] | NR* [2-4] | NR* [0.03-0.06] | ||||||
200 nmol |
20-25 | 80+ | 0.6-0.8 | 8-12 | 24+ | 0.26-0.4 | 4-6 | 8+ | 0.13-0.2 | ||||||
1 umol |
100-120 | 400+ | 3-4 | 40-50 | 90+ | 1.3-1.6 | 20-25 | 40+ | 0.6-0.8 | ||||||
2 umol |
200-240 | 800+ | 6-8 | 80-100 | 180+ | 2.5-3.1 | 40-50 | 80+ | 1.2-1.5 | ||||||
10 umol |
~1000 | ~4000 | ~30 | ~400 | ~900 | ~10 | ~200 | ~400 | ~60 | ||||||
15 umol |
~1500 | ~6000 | ~45 | ~600 | ~1350 | ~15 | ~300 | ~600 | ~90 | ||||||
Purity & Yield |
Purity is greater than 80% depending on oligo sequence and structure. Refer to coupling efficiency table for oligo length dependent purity and yield. No further purification required for PCR and sequencing applications. Gel purification recommended for oligos above 50 mer and all applications involving cloning and mutagenesis. **RPC is reverse phase purification using a cartridge; a substitue for HPLC. |
Purity 85% to 95% depending on oligo sequence and structure Yield and Purity will be lower for sequences with high GC content Not recommended for oligos longer than 35 mer. **RPC is reverse phase purification using a cartridge; a substitue for HPLC. |
Purity 98% to ~100% depending on oligo sequence and structure Yield will gradually decrease as length of oligo increases. Palindromes, hairpins and high GC content oligos and oligos containing stretches of 3 or more G's induce strong secondary structure and base stacking thus decreasing purity and yield. *NR Not Recommended |
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*a. Typical yield stated in the table is for unmodified random sequence oligos. Reduction in yield is observed with high GC content oligos and those forming strong secondary structures. b. Reduced yield is expected with modified oligos. The reduction percentage varies with modification type and number of sites. Typical reduction is 10%-20% per modified site. c. Yield of 33µg/A260 unit for DNA oligos is calculated for an ~equimolar base composition. Long stretches of a single base or homopolymers will have variable yields. Example for homopolymeric 20 mer: dA(20) = ~25ug/A260 Unit; dC(20) = ~40ug/A260 Unit; dG(20) = ~32ug/A260 Unit and dT(20) = ~37 ug/A260 Unit |
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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.
| RPC Purified** | Gel Purified | ||||||||||||||
| 30 mer oligo Typical Yield |
50 mer oligo Typical Yield |
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Scale |
A260 Units |
nmols |
mg |
A260 Units |
nmols |
mg |
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50 nmol |
~2 | ~4 | ~0.06 | ~1 | ~2 | ~0.03 | |||||||||
200 nmol |
~4 | ~8 | ~0.12 | ~2.5 | ~5 | ~0.07 | |||||||||
1 umol |
~12 | ~24 | ~0.36 | ~8 | ~16 | ~0.24 | |||||||||
2 umol |
~20 | ~40 | ~0.6 | ~15 | ~30 | ~0.45 | |||||||||
10 umol |
~100 | ~200 | ~3.0 | ~75 | ~150 | ~2.25 | |||||||||
15 umol |
~150 | ~300 | ~4.5 | ~112 | ~225 | ~3.36 | |||||||||
Purity & Yield |
Purity 80% to 90% depending on oligo sequence and structure Yield and Purity will be lower for sequences with high GC content Not recommended for oligos longer than 35 mer. **RPC is reverse phase purification using a cartridge; a substitue for HPLC. |
Purity 90% to ~98% depending on oligo sequence and structure Yield will gradually decrease as length of oligo increases. Palindromes, hairpins and high GC content oligos and oligos containing stretches of 3 or more G's induce strong secondary structure and base stacking thus decreasing purity and yield. *NR Not Recommended |
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*a. Reduction in yield is observed with high GC content oligos and those forming strong secondary structures. b. Reduced yield is expected with modified oligos. The reduction percentage varies with modification type and number of sites. Typical reduction is 10%-20% per modified site. c. Yield of 33µg/A260 unit for DNA oligos is calculated for an ~equimolar base composition. Long stretches of a single base or homopolymers will have variable yields. Example for homopolymeric 20 mer: dA(20) = ~25ug/A260 Unit; dC(20) = ~40ug/A260 Unit; dG(20) = ~32ug/A260 Unit and dT(20) = ~37 ug/A260 Unit |
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Oligo Purification