Frequently Asked Questions

Oligonucleotides are stable in aqueous solution at 25ºS temperature for 1-2 weeks at a concentration above 150 ng/μl (20 μmol). It is RECOMMENDED that oligonucleotides be stored at -20 ºS for better preservation.
IT IS NOT RECOMMENDED to frequently thaw/freeze oligonucleotides if their concentration is below 100 ng/μl (15 μmol).
Oligonucleotides with a fluorescent label should only be stored in the dark.
DO NOT allow the contact of oligonucleotides with non-sterile objects, especially when working with enzymes and aggressive media.
ATTENTION!Dilution and freezing of oligonucleotides at a concentration below 15 μmol may cause a decrease in the quality of the oligonucleotide in PCR.

For standard oligonucleotides, it is recommended to carry out:
Systematic visual inspection - the solution should be clear without visible sediment.
Spectrophotometric analysis and concentration determination.
Analysis of oligonucleotides using reverse phase or ion-pair HPLC, providing a separation profile (on demand).
PAGE electrophoresis using length markers.
For modified oligonucleotides, it is recommended to control the presence of modifiers:
For fluorescent labeled oligs, the presence of the label must be monitored by PAGE electrophoresis followed by visualization of the product under a UV lamp.
For biotin oligomes, the presence of biotin in the oligonucleotide must be monitored using both electrophoretic and colorimetric assays.
For oligs with an amino group, it is necessary to control the presence of an amino group by reaction with biotin.
For phosphate group oligols, it is necessary to control the presence of the phosphate group by hydrolysis of an aliquot of the modified oligonucleotide with alkaline phosphatase, followed by electrophoretic analysis of the reaction products.

Absorbance in O.E., molecular weight (M.W.) and volume (V) may be reported in the product passport or synthesis report. This information can be used to calculate the concentration of the sample in any desired units. If you want to know the concentration in micrograms per ml, multiply the absorbance value by 33 (extinction factor) and divide it by the volume in milliliters. Formula: microgram/milliliter = O.Е. х 33/ V (in milliliters) For example, if there is 28 O.E. in 500 microliters of water, the calculation will be as follows: 28х 33 / 0,5 = 1848 microgram/milliliter. To find out the concentration in micromoles, the obtained amount in micrograms should be divided by the molecular weight (M.W.) and the volume (V) of the sample in liters. Formula: μM = O.Е.х 33 /M.W. (g) / V (L) Если нужно знать концентрацию в микромолях на мл, расчет выполняется по формуле: 28 х 33/9000 / 0,5 = 0,205 микромоль / милилитр. For example, if the sample has 28 O.E. in 500 microliters and the molecular weight is 9,000 grams, the calculation will be: 28 x 33/9000/0.0005 = 205.33 micromoles per liter. The O.E. value has no units and is independent of the solution volume. To calculate the number of micrograms in an oligonucleotide sample, multiply the OD value by a constant called the molecular extinction coefficient. The molecular extinction factor for each oligonucleotide is slightly different, but 33 is usually used as an approximate extinction value for single-stranded DNA.

Oligonucleotides have hydroxyl groups at 3 "and 5" ends, phosphate groups do not have. But such a modification is possible.

The appearance of dried oligonucleotides may differ due to the small changes that typically occur under drying conditions. The appearance can range from a small size glass ball to a relatively large white pellet. Before opening, all sample tubes must be centrifuged to prevent loss of product that may have been dispersed on the tube walls.

The scale of the synthesis is determined by the amount of starting material (for example, a scale of 50 nanomoles means that there are 50 nanomoles of the initial base on the polymer used for the synthesis.) From the starting material, a significant proportion may not be available for use due to chemical modification or steric problems. The actual output is calculated as described above.

The length is determined by the resolution limit of the purification method and the attachment efficiency provided by the DNA synthesizer. Currently, it is possible to synthesize oligonucleotides up to 200 or more bases long and obtain sufficiently high yields by clearing PAGE for successful gene construction. Remember that the longer the length, the more likely it is to accumulate errors in the sequence.

Many of the modified amidites are unstable and have lower attachment efficiency compared to unmodified bases. All modified oligonucleotides should be purified either by RP cartridge or by HPLC to more effectively remove off-target sequences. The product is also lost by purification, but the final product, although with a lower yield, is much cleaner.

The limited stability of the reagent (less than 48 hours) and the low addition efficiency of the reagent require the use of an excessive modifying reagent to obtain a sufficient amount of product, resulting in a high cost of synthesis of modified oligonucleotides.

Yes, standard synthesis protocols provide for the synthesis of degenerate oligonucleotides.

The presence of a phosphate group at the 5 "end is required to carry out the ligation reaction. If the oligonucleotides do not contain this group at the 5 "end, no ligation will occur. The problem can be solved by phosphorylation of the oligonucleotide enzymatically with a kinase before use in ligation reactions.

Purified water, sodium phosphate buffer or any other biological buffers are acceptable as diluents. The recommended volume of diluent is 100 µL - 1 mL, the concentration depends on the application and the yield of the resulting product. The standard concentration for PCR primers is 0.1 millimole.

The final yield (amount) of the oligonucleotide can be determined through two conversion factors. The first is a nanomole per unit absorbance (O.D.). The second is micrograms per unit absorbance (O.U.) Absorbance is measured on a spectrophotometer at 260 nanometers. To find the final yield in nanomoles, multiply the absorbance of the measured oligonucleotide and the conversion rate of absorbance in the nanomoles. To find the final yield in micrograms, multiply the absorbance of the measured oligonucleotide and the conversion rate of absorbance into micrograms.

Lyophilized oligonucleotides are sufficiently stable at room temperature, at least in the short term. But in the long term, oligonucleotides need to be stored dry at -70 C.

It is not recommended to store oligonucleotides in solution at -20 ° C. Freeze and thaw cycles can lead to degradation of oligonucleotides. Freezing and thawing are acceptable when oligonucleotides are removed from the freezer and then placed back after use. Unfortunately, unintended freeze/thaw cycles may also occur. The most common causes are frost-resistant freezers and temperature fluctuations in the freezer.

The oligonucleotides may be dissolved in either sterile water or buffer. We recommend dissolving the oligonucleotides, then aliquoting into several tubes and working solutions (aliquots) should be stored at 4 ° C. The remaining aliquots should be lyophilized (dried using freeze drying) and dried samples (oligonucleotide granules) stored in a refrigerator at -70 ° C. Under these conditions, oligonucleotides in the form of dried granules can be stored for a long time. Liquid oligonucleotides are generally stable for a week when stored at 4 ° C.

With reference to oligonucleotides, the term "desalting" means the removal of small molecules of organic impurities which are by-products of synthesis. The only salt is the DNA itself. The negative charge of the phosphate skeleton of the oligonucleotide is compensated by a positively charged counterion to maintain electrical neutrality (ammonium ions are usually used). Post-synthetic treatment involves deprotection by incubation in ammonia or other basic reagent. The product can be purified to remove cleaved basic protecting groups and other synthetic by-products. This process is commonly referred to as desalination. For standard PCR reactions, oligonucleotides need not be desalted. For the kinase reaction, desalting of oligs is necessary.

The coupling efficiency of each new base (monomer) is typically 99% or more.

After the first step of attachment, the volume of dimethoxytrityl protecting group (DMT, Trityl) that appears during deblocking is directly proportional to the amount of full-length oligonucleotide produced in the previous cycle. The dimethoxytrityl protecting group to be removed has an orange color, the intensity of this color can be measured by UV spectrophotometry. By comparing the color intensity obtained after the first and last addition operations, the addition efficiency or synthesis quality can be calculated.

Phosphoamidite monomers (amidites) have a 5 '-end with a dimethoxytrityl protecting group. This monomer carrier-bound group is removed during the unlocked operation on each synthesis cycle. The final dimethoxytrityl protecting group remains or is removed, depending on the purification method chosen. If purification is carried out using an RP cartridge or HPLC, it is necessary not to remove the final protecting group, but if it is necessary to purify oligonucleotides using polyacrylamide gel electrophoresis (PAGE), then the final DMT protection must be removed.