Oligonucleotides Service

Q: How can I order?

A: To avoid copy mistakes or delay in the processing of the order, we strongly suggest to forward your sequences via our Primm On-Line Ordering System (POLOS).
You can register at www.primmbiotech.com to get the user-ID and the password by which you can enter your account.
In the on-line ordering format, it’s possible to upload an Excel file, simply assigning one column of your file to the oligo names and another one for the sequences.
In that file you can also specify the eventual different modifications, labelings or purifications.
Please, follow the instructions reported on the on-line page to set up the Excel file in the proper way.

Q: How can I cancel or modify my order?

A: It is possible to change or cancel your order within 2 hours after your order has been sent.
Please, send an email to This e-mail address is being protected from spambots. You need JavaScript enabled to view it .

Q: What kind of oligonucleotide services can Primm supply?

A:

• Standard oligos: PCRPure (salt free)
• HPLC Purification
• PAGE Purification

• Base and backbone modifications
• Non fluorescent modifications
• Dye modifications

• Modifications and labelings
• Customized concentrations and volumes
• Suspended or lyophilized

• Modifications and labelings
• Backbone modifications (i.e. 2’-OMe bases)

Q: What are Primm synthesis scales and guaranteed yields/quantities?

A:
50 nmoles scale (yields > 5 OD; > 150 ug)
0.2 umoles scale (yield > 10 OD; > 300 ug)
1 umole scale (yiled > 45 OD; > 1350 ug)
5 umoles scale (yield > 150 OD; > 4500 ug)
10 umoles scale (yield > 300 OD; > 9000 ug)

Q: Which modifications and labelings are possible?

A:
• DNA modifications (Phosphororylation)
• Base and backbone modifications (Phosphorothioated linkages; Inosine; Deoxyuridine; 2’-OMe bases; 5-Br-dG; 5-Br-dU; 5-Me-dC etc…)
• Non fluorescent modifications (Aminolinker C3, C6, C7, C12, Thiol, Biotin, Digoxigenin etc…)
• Dye Modifications (FAM/HEX/TET; Tamra/ROX; JOE; IRD 700/800; Cy3/Cy5 etc…)
• RNA modifications and labelings
Other modifications and labelings are available upon request.
Please do not hesitate to contact our customer support team ( This e-mail address is being protected from spambots. You need JavaScript enabled to view it or This e-mail address is being protected from spambots. You need JavaScript enabled to view it ).

Q: Which dye/quencher combinations are possible for dual labeled probes?

A: We offer a wide range of fluorescent reporters and different quenchers for Dual Labeled Probes and Molecular Beacons

5’ Fluorescent Reporters:

• Fluoresceine (6-FAM)
• Hexachloro-Fluoresceine (HEX)
• Tetrachloro-Fluoresceine (TET)
• Tamra
• ROX
• JOE
• Cy3
• Cy5

3’ Quenchers:

• Tamra
• Dabcyl
• Blackhole Quencher 1 (BHQ1)
• Blackhole Quencher 2 (BHQ2)
• Blackhole Quencher 3 (BHQ3)

Q: How are oligos produced at Primm labs?

A: Oligonucleotides are produced on highly automated AB 3900 DNA synthesiser, following the phosphoramidite chemistry protocol. The oligonucleotides are synthesised in 3’ to 5’ direction while attached covalently to a solid support (solid phase synthesis). The building blocks used for synthesis are DNA phosphoramidite nucleosides which are modified with different protection groups. Synthesis starts with the cleavage of the 5’ protection group dimethoxytrityl (DMT). The resulting free 5’ hydroxyl function is coupled with an activated phosphoramidite nucleoside. Typically coupling efficiency is between 98% and 99.5%. Since the coupling efficiency is not 100%, a small percentage of truncated sequences are produced at every coupling step. If these failure sequences are allowed to further react, unwanted mutants would result. This problem is overcome largely by capping the remaining free 5’ hydroxyls through acetylation. But, some molecules fail to acetylation and continue to participate in the following synthesis cycles, resulting in near full length molecules that contain internal deletions (the so-called (n-1, n-2, n-3 etc.. species). After coupling, the DNA bases are connected by an unstable phosphite trimester that it’s suddenly converted to a stable phosphotriester linkage by oxidation. The oxidation step completes one cycle of oligo synthesis. DNA synthesis can continue with the removal of the DMT group at the 5’-end of the growing chain, starting another cycle of nucleotide addition.

Q: Does the oligo sequence influence the synthesis?

A: Due to our 20-years experience in oligonucleotide synthesis, we are able to predict those sequences that are suppose to be difficult to produce. For example, oligonucleotides with a high percentage of "G" residues, with stretches of “CG” or stretches with repetition of the same base are probably difficult to synthesise. Oligos with self-complementary sequences tend to form aggregates, and this formation can complicate HPLC and PAGE purification.

Q: How do I select the purification method that best fits to my experiment?

A: When starting an experiment it’s fundamental the selection of the correct purification option, taking into consideration the intended application. In DNA synthesis, each oligonucleotide is coupled sequentially to the growing chain in a 3’ to 5’ direction. In each coupling cycle, a small percentage of the oligo chain will not be extended, resulting in a mixture of full-length product (n) and truncated sequences (n-1; n-2; n-3 etc…).
With an appropriate purification is possible to separate the full-length product from the truncated sequences and the purity level required depends on the potential problems that the presence of truncated oligomers may cause.

For some applications, it is crucial that the majority of the oligo is represented by the full-length (n); for others, a small presence of shorter oligos (n-1, n-2, …) will not affect the experimental results.

PCRPure – Desalted oligos (Gelfiltrated on Sephadex)
Every oligo manufactured by Primm is desalted. The desalting procedure removes residual by-products from the synthesis, cleavage and deprotection procedures.
This type of purification is acceptable for PCR and sequencing and, for oligos less than 40 mer, it’s also proper to RT-PCR and to backbone modified oligos.

Reverse-Phase HPLC (RP-HPLC)
Reverse-Phase HPLC is an effi cient purifi cation method for labeled oligos, as the intrinsic lipophilicity of the dye/fluorophore provides excellent separation of the labeld oligo from the not labeld.
Furthermore, RP-HPLC is a method of choice for larger scales (> 1 umole) due to the capacity and resolving properties of the column. The resolution (based above all on the lipophilicity) will decrease with the length of the oligo, therefore, RP-HPLC is usually not recommended for oligos longer than 50 bases.

PAGE
The basis of the PAGE separation is charge over molecular weight, leading to excellent size resolution, resulting in purity levels of 95–99% full-length product. Yields from PAGE are lower than from other methods due to the complex procedure required for extracting oligos from the gel and the removal of the vast majority of truncated products. This technique is recommended when a highly purified product is required (i.e.: annealing, cloning, mutagenesis, gene synthesis, crystallography, NMR etc…). Moreover PAGE is recommended for longer oligos (≥50 bases).

Q: Which quality controls are performed at Primm before the oligos delivery?

A:
Trityl Monitoring
The trityl monitoring allows the extimation of the coupling efficiency during the synthesis. It consists in the measure of the absorbance of the trityl cation produced during the coupling of each base. The absorbance of the trityl cation is UV determined at 498 nm.

OD Measurement
The final yield of an oligo is determined by measuring the OD (Optical Density) values. One OD260 unit of DNA is the amount of DNA that gives an absorbance reading of 1.0 at a wavelength of 260 nm, for a sample dissolved in 1.0 ml total volume of ddH20 which is read in a 1 cm quartz cuvette. 1 OD260 corresponds to approx. 33 μg/ml of single stranded DNA, depending on the GC content.

MALDI-TOF MS and PAGE controls
To ensure the identity and qualitative purity of Primm oligonucleotides, we perform MALDI-Tof and PAGE analysis at each daily run of synthesis on randomly selected oligos. These cross controls enable us to observe the general production trend and to be confident in the quality of our oligos.

Q: How long are oligos stable and how should I store them?

A: We usually supply oligonucleotides in a lyophilised state, since this form is less sensitive to degradation by nucleases and more stable for transportation.
We recommend storage of the lyophilised material at room temperature for 3 months or at -20 °C for 6 months. Once you have dissolved the oligonucleotides, in sterile water or in a buffer, the best way to store them is to prepare aliquots of several tubes and to store them at -20 °C or lower for 3/6 months. The sample which is currently in use can be kept in a refrigerator at +4° C for a short time to avoid continuous freezing and thawing of the solution.

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