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| Oligonucleotides : |
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| Q1 、How should I store my oligo and how long will it last? |
| Oligonucleotides are chemically stable. Left dry, they should be good for years. Once hydrated, they are susceptible to degradation by nucleases. But even so, hydrated oligos, if handled correctly, should still be stable for years. |
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| Eurogentec supply the majority of our oligonucleotides dry, since this form is less sensitive to degradation by nucleases and dried oligos are also stable at room temperature for transportation. |
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| For short term use (1-2 weeks) you can store your oligos at 4 ℃ . For long term storage we recommend to store the oligos dry or in solution at -20 ℃ . or below. If an oligonucleotide is to be used repeatedly over a long period of time we recommend that you aliquot it in nuclease-free, sterile water or buffered solutions containing EDTA into several tubes for storage at -20 ℃ .or lower. No account should oligonucleotides be repeatedly frozen and thawed as this process can lead to physical degradation of the oligos. Fluorescently labeled oligos should be stored in light-free conditions.When handled in this way Eurogentec customers have used the same sample for periods in excess of 5 years. |
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| Q2 、How do I calculate the extinction coefficient of an oligonucleotide? |
The following calculation can be used to determine extinction coefficients of oligonucleotides. [((nA x 15.4) + (nG x 11.7) + (nC x 7.3) + (nT x 8.8) + (nD x 12.0) + (nM x 11.3) + (nH x 10.4) + (nW x 12.1) + (nR x 13.6) + (nY x 8.0) + (nV x 11.4) + (nS x 9.6) + (nK x 10.7) + (nN x 10.8) + (nB x 9.2))] x 0.9 The factor of 0.9 at the end of the calculation is to allow for base stacking within a single strand. For double stranded DNA a factor of 0.8 rather than 0.9 should be used.
In the above equation, D=A/G/T, M=A/C, H=A/C/T, W=A/T, R=A/G, Y=C/T, V=A/C/G, S=C/G , K=G/T , N=A/G/C/T, B=C/G/T |
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| Q3 、 How do I calculate the molecular weight of an oligonucleotide? |
| The following formula can be used to calculate the molecular weight of DNA oligonucleotides: M {g/mol}= (nA x 249.086189) + (nG x 265.081104) + (nC x 225.074955) + (nT x 240.074621) + (nD x 251.41397133) + (nM x 237.080572) + (nH x 238.07858833) + (nW x 244.580405) + (nR x 257.0836465) + (nY x 232.9120855) + (nV x 246.41408267) +(nS x 245.0780295) + (nK x 252.5778625) + (nN x 244.82921725) + (nB x 243.41022667) + (63.971417) x(Length - 1)) + 2.015650 Where D=A/G/T, M=A/C, H=A/C/T, W=A/T, R=A/G, Y=C/T, V=A/C/G, S=C/G , K=G/T , N=A/G/C/T, B=C/G/T |
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| Q4 、 How do I calculate the melting temperature (Tm) of an oligonucleotide? |
The melting temperature of an oligonucleotide hybridized to a DNA target can be calculated from the following equation:
Sequence with less than 20 bases:
· Tm{ 皚 = 2 (nA + nT) + 4 (nG + nC) Sequence with 20 or more bases:
· Tm=81.5 + 16.6log[Na+] + 0.41(%G + %C) - 675/length With [Na+]=0, 05M and length = length of the oligo
· For degenerated oligos, the (%G + %C) value must be the lowest one
· For oligos with Inosine, length = (Length of the oligo)
· (Number of Inosine bases) |
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| Q5 、 What does 1 OD stand for? |
| 1 OD260 (optical density) unit is defined as the amount of oligonucleotide which, when dissolved in a volume of 1.0 ml, results in an absorbance of 1.0 when measured at 260 nm in a 1 cm path-length quartz cuvette. 1 OD260 unit corresponds to approx. 33 ug of single strand DNA. |
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| These relationships, however, can be inaccurate for short fragments of DNA, such as oligonucleotides. Base composition and even linear sequence will affect optical absorbance. Hence the precise value of the OD to mass relationship is unique for each oligo. |
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| For example 1.0 OD260 of CCCCCCCCCC (10 mers) equals 39 ul whereas 1.0 OD260 of AAAAAAAAAA (10 mers) equals only 20 ug. |
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| The extinction coefficient, E260, defines the relationship between mass and optical absorbance and can be calculated for any sequence. |
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| The following equation defines a shortcut to calculate the number of nanomoles present given an OD reading and extinction coefficient: Nanomoles = ( OD260 / E 260 ) * 10 6 . Example: 1.0 OD260 units of primer M13 Forward, 5' GTA AAA CGA CGG CCA GTG 3'; Molar extinction coefficient (E 260 ) = 182,800 L / mole * cm; Nanomoles = ( 1.0 / 182,800 ) * 10 6 = 5.47 nmole. Converting Nanomoles to Milligrams - The following equation defines the relationship between nanomoles and milligrams: Milligrams = Molecular Weight * Nanomoles * 10-6: Example: 1.0 OD260 units of primer M13 Forward, 5' GTA AAA CGA CGG CCA GTG 3' - Molecular Weight = 5558.7; Milligrams = 5558.7 * 5.47 * 10-6 = 0.0304 mg = 30.4 microgram. The molecular weight of each oligonucleotide is calculated according to its sequence and is provided on the specification sheet. |
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| Q6 、 HPLC purification, is it better than cartridge purification? |
| HPLC purification produces higher yields of purified oligonucleotides than cartridge purification. It gives additional feedback which simple cartridge purification cannot do by providing our skilled technicians with a detailed chromatagram which reveals the purity of the oligonucleotide and indicates if there are any problems which were not picked up in the trityl analysis (synthesis quality analysis). Some impurities in modified oligonucleotides are not removed by cartridge purification (e.g. a common impurity in HEX-labelled oligonucleotides). |
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| Q7 、 How does Generay quantify its oligos? |
| We carefully measure the O.D. value for your custom oligonucleotide by measuring the absorption at 260 nm using an UV spectrometer, and we provide this information on the oligonucleotide specification sheet as: the number of OD260 units, the number of nanomoles and the number of milligrams. |
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| The amount of oligo present in milligrams and nanomoles is calculated from the OD measurement. The following equation defines a shortcut to calculate the number of nanomoles present given an OD reading and extinction coefficient: Nanomoles = ( OD260 / E260) * 106. Example: 1.0 OD260 units of primer M13 Forward, 5' GTA AAA CGA CGG CCA GTG 3'; Molar extinction coefficient (E260) = 182,800 L / mole * cm; Nanomoles = ( 1.0 / 182,800 ) * 106 = 5.47 nmole. Converting Nanomoles to Milligrams - The following equation defines the relationship between nanomoles and milligrams: Milligrams = Molecular Weight * Nanomoles * 10-6: Example: 1.0 OD260 units of primer M13 Forward, 5' GTA AAA CGA CGG CCA GTG 3' - Molecular Weight = 5558.7; Milligrams = 5558.7 * 5.47 * 10-6 = 0.0304 mg = 30.4 microgram. The molecular weight of each oligonucleotide is calculated according to its sequence and is provided on the specification sheet. |
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| Q8 、 What is a synthesis scale? |
| The synthesis scale is based on the amount of the first base attached to the solid support (controlled-pore glass, CPG) to start the oligo synthesis, not the amount of the final material synthesized. For larger scales, the amount of solid support is increased. The scale does not indicate the expected yield. Losses in yield may occur during synthesis, post-synthetic processing, transfer of material and quality control. The quantity of DNA ultimately received is usually lower than the theoretical yield. For example, a 20 mer synthesized at a 200 nmole scale will produce approximately 80 nmoles. |
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