Oligo Configuration Options
Oligo Purity Selection Guide
Many different applications demand different purity or scale to work well when it comes to custom DNA oligo synthesis. Here are some guidelines for purity and synthesis scale selection for different applications.
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
(40 nm, 7-30 bp; 50 nm-1 µm, 7-100 bp)
|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.|
(50 nm-1 µm, 7-100 bp)
|Based on reverse phase chromatography; removes failure sequences from the completed synthesis.||Provides full-length sequences needed in some applications|
(50 nm-1 µm, 7-65 bp)
|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).|
(50 nm, 20-100 bp; 200nm-1 µm, 7-100bp)
|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.|
|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 has 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 for Invitrogen 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.|
|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 have a different modification from the options below.
|Modification Name||Electronic Ordering Code||Absorption Maximum (nm)||Emission Maximum (nm)||Extinction Coefficient (OD/mole) At 260 nm|
|Fluorescent Dye Modifications|
|Molecular Probe Dyes|
|Dye||Electronic Ordering Code||Color||Absorption Maximum (nm)||Molar Emission Maximum (nm)||Extinction Coefficient (cm-1M-1)|
|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|
|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|
|RHODAMINE GREEN™ Dye*||RGA||Green||504||532||78,000|
|RHODOL GREEN™ Dye*||RGB||Green||496||523||63,000|
|TEXAS RED® -X*||TRX||Red||583||603||136,000|
*Red colored dyes are HPLC purified.
|Alternative bases||Description||Electronic Ordering Code|
|Deoxyuracil||Useful with UDG for ligase-free cloning.||U|
|Deoxyinosine||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, F.H., Castro, M.M., Aboul-ela, F., and Tinoco, I. Jr. (1985) Nucleic Acids Res.13, 8927.)||I|
|Phosphothiates||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.||(see below)|
|Mixed Bases*||Degenerate bases. Equal amounts of the designated bases are delivered by the synthesizer.|
*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.
|Dye Name||Absorption Maximum (nm)||Emission Maximum (nm)||Extinction Coefficient (OD/mole) At 260 nm||Electronic Ordering Code|
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.
Custom Oligo Modification Services
Bringing you more
We are proud to offer an enhanced selection of custom oligonucleotide options that do not appear on our standard modification menus.
Each order is treated as a special project by our dedicated services team. Upon completion, the project is reviewed by our Technical, Quality, and Manufacturing teams to deliver the best the industry has to offer.
Frequently Asked Questions
A. Our synthesizers allow us to use any commercially available modification and add them to your oligo. We also offer post-synthesis labeling with fluorescent dyes such as our superior Alexa Fluor® dyes.
Q. What scales are available?
A. We can manufacture from 200 nmol to 1 gram scales in a variety of purities. Just let us know what you need.
Q. What purity selections do you offer?
A. We offer standard desalted, HPLC, and in vivo purifications.
Q. Do you offer custom siRNA libraries?
A. We can offer plated sets of siRNAs designed to your list of favorite targets. Contact us today for a quote.
For more information, call 800.955.6288 x2, or email us at email@example.com.
For Research Use Only. Not for use in diagnostic procedures.