How to store the DNA primers
Normally, oligos should be stable at -20°C and can be stored at that temperature for more than a year. Although stable in solution, oligos will be degrade if the storage solution is contaminated with nucleases. Therefore, we recommend that oligos be stored in the dried form. If you want to store oligos in solution, it is best to aliquot the oligo into several tubes and store them separately. Oligos can also be subject to degradation due to the 'Freezing and Thawing Effect' when the oligo solutions are frozen and thawed repeatedly.
Choice of Purity Level
For some applications, such as PCR or sequencing, standard desalting is perfectly fine because the truncations and deletions will not affect your results appreciably.
For other applications, such as cloning, mutagenesis, and gel shift, full length product is of utmost importance, PAGE purification should be strongly considered. PAGE will result in the highest purity level in terms of full length product, and your oligos will be at least 90% full length. PAGE purification does tend to result in lower yields than HPLC purification, however.
If you need a relatively clean product, but also need a higher yield, you should consider standard HPLC for oligos up to 50nts in length, and HPLC for longer oligos. HPLC will result in a 90% purity level for longer oligos. You should also take the types of modifications on your oligo into account when choosing a purification method.
PAGE purification can damage certain modifications, including many fluorophores and some modifications used for attachment. PAGE purification should be avoided for the following modifications: Any fluorophore, Acrydite, Amino modifiers, Biotin, Digoxigenin, I-Linker, Spacer 18, Thiol modifiers. HPLC would be the purification of choice in this case.
Calculation of Tm
Tm (melting temperature) refers to the temperature where 50% of oligonucleotides exist in duplex form and the rest in single-strand form. The estimation method based on the GC content for long sequences is :
Another easier way to calculate the Tm is to use following equation:
If the experiment does not yield anticipated results, it is recommended to lower the annealing temperature by 4-5 degrees from the Tm value. If there are many non-specific products, trial-and-error approach should be taken to obtain the optimum annealing temperature.
Calculation of MW
The molecular weight of an oligo can be calculated with the following equation:
Measurement of OD and Concentration (µM, ng/µL)
The quantity of oligos is often described in O.D. units which actually express light absorbance. One O.D. corresponds to the amount of oligo in a 1mL volume that results in an optical density of 1 in a 1cm path-length cuvette. This corresponds to approximately 33 µg of oligo, although it varies for each particular oligo depending on its sequence. The concentration of an oligo of known sequence can be calculated since it is known that the extinction coefficient (in a 1cm path-length cuvette) for each of the bases at 260 nm is
For any given oligo, multiply the number of times each base appears by its extinction coefficient. Then add the resulting four numbers to obtain the extinction coefficient (e) for the entire oligo. The concentration (C) can then be calculated from the equation (for a 1cm path-length cuvette):
Coupling efficiency is the major factor affecting the length of DNA that can be synthesized. Base composition and synthesis scales will also be contributing factors. Table 1 shows that at 99% coupling efficiency, a crude solution of synthesized 95-mers would contain 38% full-length product and 62% (nx) failure sequences. This is before other chemical effects have been taken into account such as depurination. Depurination mainly affects the base A. The frequency of depurination is small but will increase significantly with primer length. For these reasons, we specify a maximum length of 100 bases, which we believe is the maximum length that can be synthesized routinely and economically.
Table 1. How coupling efficiency affect purity of synthesized oligos
|No. of bases|
|99% Coupling||98% Coupling|
Purification of the Oligo
For the applications such as cloning, site directed mutagenesis or quantitative gene detection, oligos of higher purity are preferred to get the satisfactory results. Since desalted oligos might not be sufficient in such cases, HPLC purification has been commonly used for that purpose.
HPLC with an RP column delivers 90~98% purification efficiency and is adequate for purification of oligos up to 35-mer.
For the purification of long-length oligos (up to 100-mer), ValueGene recommends PAGE purification which uses cross-linked polyacrylamide gel as purification matrix. Though PAGE shows high purification efficiency, it has a couple of shortcomings in that extra processes such as extraction and desalting are required following PAGE and subsequently results in a decrease in purification yields.
Choice of Scale
Synthesis scales refer to the amount of starting material present, not the amount of final product produced. This is the same for all manufacturers of synthetic DNA. When a 50 nmole scale synthesis is specified, approximately 50 nmoles of the first base is added to the DNA synthesizer. For an average 25-mer, at least 25% of this starting material will result in failure sequences, hence it is not possible to produce 50 nmoles of full-length product from a 50 nmole scale synthesis.
Please refer to our product specification section for the details about the quantity we offer at each scale level.