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

Purification Method Description Benefit
(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.


Application-Purity Guide

Application Suggested Purity
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.

Modification Options

Please email us if you have a different modification from the options below.


5' Modifications


Modification Name Electronic Ordering Code Absorption Maximum (nm) Emission Maximum (nm) Extinction Coefficient (OD/mole) At 260 nm
Generic Modifications
Aldehyde ALD n/a n/a n/a
Amine® (Primary) AMN n/a n/a n/a
Biotin BIO n/a n/a n/a
Phosphate PHO n/a n/a n/a
Thiol THL n/a n/a n/a
Fluorescent Dye Modifications
Fluorescein FLO 494 520 20,900
FAM FAM 494 520 20,900
HEX HEX 535 553 31,580
ROX* ROX 576 601 22,500
TET TET 522 538 16,255
TAMRA* TAM 565 580 32,300


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
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
RHODAMINE GREEN™ Dye* RGA Green 504 532 78,000
RHODAMINE GREEN™-X* RGC Green 503 528 74,000
RHODOL GREEN™ Dye* RGB Green 496 523 63,000
RHODAMINE RED™-X* RRA Red 560 580 129,000
TEXAS RED®* TXR Red 595 615 80,000
TEXAS RED® -X* TRX Red 583 603 136,000

*Red colored dyes are HPLC purified.



Internal Modifications


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)
A-Phosphorothioate   F
C-Phosphorothioate   O
G-Phosphorothioate   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

Dye Name Absorption Maximum (nm) Emission Maximum (nm) Extinction Coefficient (OD/mole) At 260 nm Electronic Ordering Code
Biotin n/a n/a n/a BIO
Phosphate n/a n/a n/a PHO
Amine® n/a n/a n/a AMN
Thiol n/a n/a n/a THL

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


Off-the-Menu Modifications

Do you need to order a DNA or RNA oligo with a special modification but can’t find it on our Custom Oligo pages? Then order them here at Custom Oligo 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

Q. What modifications are available?
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.
Start your project now! Click here to get our order form and begin the process. A project manager will respond to your inquiry within 2 business days.

For more information, call 800.955.6288 x2, or email us at custom.services@lifetech.com.