Calculations / Conversion Factors
What is the definition of OD260?
One OD unit of DNA is the amount of DNA that gives an absorbance reading of 1.0 at 260nm for a sample dissolved in 1.0 ml total volume of ddH20 which is read in 1 cm quartz cuvette. (OD = "Optical Density").
For Oligos, 1 OD is approximate 33µg of DNA.
How is Melting Temperature (Tm) estimated for Oligos ?
The melting temperature (Tm value) of an Oligo depends on the length of the sequence, the G+C content of the sequence, and the type and concentration of present cations, particularly sodium ions (Na+).
A variety of formulas have been used for predicting Tm values. These tend to be fairly accurate for long DNA sequences, but are less exact for Oligos.
The following formula is recommended for Oligos ranging in length from 20 to 100 residues, and [Na+] concentrations ranging from 0.01 M to 1.0 M:
Tm (in degree C) = 81.5 + 0.41 (G%+C%) + 16.6 log[Na+] - 500/length
For example, for Oligos with a 55% G + C content in a 0.1 M Nacl solution:
Tm | =81.5 + 0.41(55) + 16.6 log [0.1] - 500/length |
| =81.5 + 22.5 - 16.6 - 500/length |
| =87.5 -500/length |
eg | 20mer: 62°C; 25mer: 67°C; 30mer: 71°C; |
| 35mer: 73°C |
How can I calculate the annealing Temperature for PCR?
Calculate the melting temperature as above and subtract 5 to 10 °C
If the two Oligos have different melting temperatures, do NOT average the numbers. Use the lower number so that both of the Oligos can anneal.
eg | 20mer: 52-57°C; 25mer: 57-62°C; 30mer: 60-65°C; 35mer: 63-68°C) |
Molecular Weight
How is the molecular Weight of an Oligo calculated?
MW | = (dA x 313.1) + (dC x 289.1) + (dG x 329.1) + (dT x 304.2) - 61 |
where dA, dC, dG, dT are numbers of dA's, dC's, dG's and dT's in an Oligo and the 5' and 3' ends contain free hydroxyl groups. Add 78 to the MW for 5'phosphorylated Oligos.
What are the approx. conversion factors?
1 OD260 unit of an Oligo | ~ 33µg |
1 OD260 unit of ss RNA | = 40µg |
1 OD260 unit of ds DNA | = 50µg |
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1µg of 1kb DNA | = 1.62 pmol (3.24 pmoles of ends) |
1 µg of pBR322 DNA | = 0.36 pmol DNA |
nmoles of Oligo = OD x 100/length of Oligo (rule of thumb)
nmoles of Oligo= OD x 1000/millimolar extinction coefficient
Scale
What is the synthesis scale?
The scale of synthesis is the amount of 3'base that we start the synthesis with. It is NOT the expected final yield. The yield depends on the size of the Oligonucleotide, the coupling efficiency, and the base composition.
What is the theoretical yield?
The theoretical yield of the full length product can be determined using the formula 0.985n, wherein 0.985 represents the coupling efficiency and n the length of the Oligo. Hence, the theoretical yield of the full length product of a 50mer is 47% at a coupling rate of 0.985. Truncated Oligos are not eliminated by desalting but by HPLC or PAGE purification.
How many reactions can I carry out with my Oligo?
In PCR, the Genomic scale yields approx. 700-3500 reactions, the 0.04µmol scale approx. 1000 to 5000 reactions and the 0.2µmol scale 4000 to 20000 reactions.
Purity
What does Desalting of an Oligo mean?
All our Oligos are at least desalted. Desalting is necessary to remove protecting groups (which prevent unwanted side reactions during synthesis) and residual salts which can affect the biological activity of the Oligos. Desalting is especially important for standard sequencing and in vitro mutagenesis.
How do I know which purification grade is sufficient?
The purity required for a specific application depends on the potential problems which would result from the presence of failure sequences (n-1, n-2,....Oligomers).
Please read the recommendations in this catalogue. If you intend to use an Oligo for a high risk project or in routine diagnostic purpose, we recommend purified Oligos.
Mutations in Oligonucleotides
The following text informs our customers about a problem most Oligo companies do not like to talk about.
All Oligo companies experience mutations in their Oligos.
One reason for mutations may be regions in Oligo sequences which, because of their folding properties, are very difficult to synthesize and therefore have an increased mutation rate at precisely these points.
Other reasons are limitations in chemistry and synthesis technology. These are not mistakes during manual typing of sequences (sequences are imported electronically and error free from the web-server into the production software), but the errors affect only a small portion of the molecules and are spread randomly over the sequence at a very low rate. We know that mutations are very frustrating for our customers, therefore we invest a lot of time and money in research on reducing the mutation rate.
The mutation rate is not a problem for methods like sequencing, hybridization, gel shift etc., because the mutation background is too low. Very rarely frustration may arise when Oligos are used for cloning projects because mutations in Oligos may become visible. In this case it is important to sequence further clones because there is a good chance that you will find the correct clone.
The mutation rate can be determined by various methods. The standard procedure which we have used successfully for many years is:
We first assemble a synthetic 145bp fragment with 2 long, overlapping 80mer Oligos. After PCR amplification with a high fidelity polymerase (Pfu I), the construct is cloned and sequenced. 96 clones are sequenced and the faulty bases are counted. Two values are calculated: The number of mutations per clone (mutation rate) and the percentage of correct clones.
As we want to constantly insure high quality,we periodically perform this test on all synthesizers. Over the last 3 years, our results have shown that the average percentage of correct clones was > 77% for a 145bp fragment.
