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Oligo Configuration Options
Our oligos are available in a range of synthesis scales, purification options, and modifications. Many different applications demand different scale or purity to work well when it comes to custom DNA oligo synthesis. Learn more here about how to choose the best oligo configuration for your applications.
To assist in the selection process, we have compiled a list of available options and guidelines below:
Synthesis scale for PCR applications
When ordering custom oligos for PCR applications, the scale of synthesis determines the number of reactions provided. The table below assumes a 100 µl PCR reaction and a final oligo concentration of 0.1 to 0.5 µM.
| Scale of synthesis | Estimated number of reactions |
|---|---|
| 25 nmole | 500 to 2,500 |
| 50 nmole | 1,000 to 5,000 |
| 200 nmole | 4,000 to 20,000 |
| 1 µmole | 20,000 to 100,000 |
| 10 µmole | 100,000 to 1,000,000 |
Understanding why oligos sometimes require purification
Following DNA synthesis, the completed DNA chain is released from the solid support by incubation in basic solutions such as ammonium hydroxide. This solution contains the required full-length oligo but also contains all of the DNA chains that were aborted during synthesis (failure sequences). If a 20-mer was synthesized, the solution would also contain 19 mer failures, 18 mer failures, 17 mer failures etc. The amount of failure sequences present is influenced by the coupling efficiency. These failure sequences can compete with the full-length product in some applications such as PCR, and may therefore need removing before the oligo can be used successfully.
Purification options offered
| Purification method | Description | Benefit |
|---|---|---|
| Desalt (25 nm: 10–100 nt; 50 nm: 5–100 nt) | Oligos are processed through normal phase chromatography column which removes salts but not failure sequences. | A salt-free DNA solution, ready-to-use; suitable for many PCR and sequencing applications without further purification. |
| Cartridge (50 nm–1 µm, 7–55 nt) | Based on reverse phase chromatography; removes failure sequences from the completed synthesis. | Provides full-length sequences needed in some applications. |
| HPLC (50 nm+, 10–55 nt) | Reverse Phase High Performance Liquid Chromatography (HPLC) removes failure sequences or unincorporated label the same way as cartridge purification. | Guarantees highly purified primer required in some applications (>=85% full length). |
| PAGE (200 nm+, 7–100 nt) | Method used to differentiate full-length product from failure sequences based on size and conformation. | Provides the highest percentage of full-length oligos (>=85%) required for certain demanding applications such as mutagenesis or adapter production. |
Purity guide by application
| Application | Suggested purification option |
|---|---|
| AFLP analysis | Desalted oligos have been used successfully for Amplification Restriction Fragment Polymorphisms. |
| Antisense | HPLC-purified oligos are cited most frequently in references for antisense studies. See Minimum Yields chart for HPLC purification yields. |
| First-strand cDNA synthesis for generation of libraries | Generally oligos for first strand cDNA synthesis for library construction have some sequence at the end which codes for 5´ restriction endonuclease cloning sites. Therefore, it is best to use full-length, Cartridge, HPLC, or PAGE-purified oligos. |
| Fluorescent sequencing | All four purity grades have worked successfully forour scientists. |
| Gel shift assays | Cartridge, HPLC, and PAGE-purified oligos are recommended for gel shift assays, so as to have a homogeneous population of DNA fragments. |
| GENETRAPPER screening | PAGE-purified oligos are recommended. Primers should be phenol extracted and ethanol precipitated prior to use in the tailing reaction in GeneTrapper System. If desalted purity oligos are purchased they can be PAGE-purified using the PAGE purification protocol. |
| Isothermal sequencing | Desalted oligos are sufficient for this application, along with Cartridge, HPLC, and PAGE-purified oligs. |
| Microarrays | Standard desalted oligos are sufficient for printing onto arrays. |
| PCR | Desalted oligos work fine for standard PCR. Higher purity options will also work. |
| PCR using oligos with critical 5´ sequences (e.g., restriction endonuclease sites, RNA polymerase promoters) | Cartridge, HPLC, and PAGE-purified oligos are best for the greatest efficiency. Since oligos are synthesized 3´ to 5´, incomplete oligos (n-x oligos) will be missing the 5´ sequence. It is important to use full-length oligos that have the 5´ sequence present, otherwise there will be a population of PCR products missing the sequence intended to be installed before PCR. |
| Production of cloning adapters | Full-length oligos work best for efficient cloning. Utilize cartridge, HPLC, or PAGE-purified oligos for full length. |
| Site-directed mutagenesis | Full-length (e.g., Cartridge, HPLC, and PAGE-purified) oligos as a rule tend to give the highest percentage of mutagenized clones (especially if the intended mutation is close to the 5´ end of the oligo). Desired mutations have been obtained using desalted oligos. However, some wild-type parental vector clones tend to carry over. |
Please email us if you need a different modification from the options below.
5′ Modifications
Review our selection of generic modifications, fluorescent dye modifications, and Molecular Probes dyes.
NOTE: Modification names with an asterisk (*) indicate products that are HPLC purified.
| Electronic ordering code | Absorption maximum (nm) | Emission maximum (nm) | Extinction coefficient (OD/mole) at
| |
|---|---|---|---|---|
| Generic modifications | ||||
| Aldehyde | ALD | NA | NA | NA |
| Amine (Primary) | AMN | NA | NA | NA |
| Biotin | BIO | NA | NA | NA |
| Phosphate | PHO | NA | NA | NA |
| Thiol | THL | NA | NA | NA |
| Fluorescent dye modifications | ||||
| Fluorescein | FLO | 494 | 520 | 31,500 |
| HEX | HEX | 535 | 553 | 31,580 |
| ROX* | ROX | 576 | 601 | 22,500 |
| TET | TET | 522 | 538 | 16,255 |
| TAMRA* | TAM | 565 | 580 | 32,300 |
| *These products are HPLC purified | ||||
| Molecular Probes dyes | |||||
|---|---|---|---|---|---|
| Dye* | Electronic ordering code | Color | Absorption maximum (nm) | Molar emission maximum (nm) | Extinction coefficient
|
| Alexa Fluor 488 | 488 | Green | 490 | 519 | 71,000 |
| Alexa Fluor 532 | 532 | Green | 532 | 553 | 81,000 |
| Alexa Fluor 546 | 546 | Yellow | 556 | 573 | 104,000 |
| Alexa Fluor 555 | 555 | Orange/yellow | 555 | 565 | 150,000 |
| Alexa Fluor 594 | 594 | Orange | 590 | 617 | 73,000 |
| Alexa Fluor 647 | 647 | Red | 650 | 655 | 239,000 |
| Alexa Fluor 660 | 660 | Red | 663 | 690 | 132,000 |
| Alexa Fluor 750 | 750 | Purple | 749 | 755 | 240,000 |
| Bodipy FL | BDA | Far Red | 502 | 510 | 82,000 |
| Bodipy 530/550 | BDB | — | 534 | 551 | 77,000 |
| Bodipy 493/503 | BDC | — | 500 | 509 | 79,000 |
| Bodipy 558/569 | BDE | — | 559 | 568 | 97,000 |
| Bodipy 564/570 | BDF | — | 563 | 569 | 142,000 |
| Bodipy 576/589 | BDG | — | 575 | 588 | 83,000 |
| Bodipy 581/591 | BDH | — | 581 | 591 | 136,000 |
| Bodipy FL-X | BDI | — | 504 | 510 | 85,000 |
| Bodipy TR-X | BDJ | — | 588 | 616 | 68,000 |
| Bodipy TMR | BDK | — | 544 | 570 | 56,000 |
| Bodipy R6G | BDL | — | 528 | 547 | 70,000 |
| Bodipy R6G-X | BDM | — | 529 | 547 | 73,000 |
| Bodipy 630/650 | BDN | — | 625 | 640 | 101,000 |
| Bodipy 650/665 | BDP | — | 651 | 660 | 100,000 |
| Cascade Blue Dye | CSB | Blue | 400 | 420 | 28,000 |
| Marina Blue Dye | MNB | Blue | 362 | 459 | 19,000 |
| Oregon Green 514 | OGB | Green | 506 | 526 | 85,000 |
| Oregon Green 488 | OGC | Green | 495 | 521 | 76,000 |
| Oregon Green 488-X | OGD | Green | 494 | 517 | 84,000 |
| PACIFIC BLUE Dye | PFB | Blue | 416 | 451 | 36,000 |
| RGA | Green | 504 | 532 | 78,000 | |
| Rhodol Green Dye | RGB | Green | 496 | 523 | 63,000 |
| Rhodamine Green-X | RGC | Green | 503 | 528 | 74,000 |
| Rhodamine Red-X | RRA | Red | 560 | 580 | 129,000 |
| Texas Red-X | TRX | Red | 583 | 603 | 136,000 |
| *These products are HPLC purified | |||||
Internal Modifications
Review the list of available internal modifications and their electronic ordering codes.
| Alternative bases | Electronic ordering code | Description |
|---|---|---|
| Deoxyuracil | U | Useful with UDG for ligase-free cloning. |
| Deoxyinosine | I | Deoxyinosine has the capacity to base-pair with all four bases; however, it does so with varying affinities. The order of stabilities for the different combinations, from greatest to least stable, reported by Martin et al. are as follows: I:C, I:A, I:T, and I:G. I:C pairs were found to be slightly less stable than A:T pairs (Martin FH, Castro MM et al. (1985) Nucleic Acids Res.13: 8927.) |
| Phosphothiates | (see below) | A sulfur is substituted for one of the oxygens in the phosphodiester bonds between the nucleotides. This linkage is to the 3′ side of the designated base. |
| F | ||
| C-Phosphorothioate | O | |
| E | ||
| T-Phosphorothioate | Z | |
| Mixed bases* | Degenerate bases—Equal amounts of the designated bases are delivered by the synthesizer | |
| A+C+G+T | N | |
| A+C+G | V | |
| A+T+G | D | |
| T+C+G | B | |
| A+T+C | H | |
| A+T | W | |
| C+G | S | |
| T+G | K | |
| A+C | M | |
| C+T | Y | |
| A+G | R | |
| *Mixed bases may be designated at any position for the 50 nmole scale and above. For the 25 nmole scale, mixed bases may be designated for any except the 3'-most base. Mixed bases are achieved by having the synthesizer deliver an equal amount of each base at the given base addition. Differences in coupling efficiency may result in the end product being slightly skewed toward the base that couples with the highest efficiency. | ||
3′ Modifications
NOTE: Not all modifications are available at all scales and purifications. Modifications other than internal mixed bases are not available for oligos ordered for Next-Day delivery. Please contact technical support for more information.
| Dye name | Electronic ordering code | Absorption maximum (nm) | Emission maximum (nm) | Extinction coefficient (OD/mole) At 260 nm |
|---|---|---|---|---|
| Biotin | BIO | NA | NA | NA |
| Phosphate | PHO | NA | NA | NA |
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