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Tips and tricks

Technical information

Oligonucleotides quantification



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 approximately 33 μg of single strand DNA. 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 oligonucleotide.

Example: 1.0 OD260 of CCCCCCCCCC (10 bases) equals 39 μg
whereas 1.0 OD260 of AAAAAAAAAA (10 bases) equals only 20 μg

We carefully measure the OD value for your Custom Oligonucleotide by measuring the absorption at 260 nm using UV spectrophotometer. This information is provided on the oligonucleotide Technical Data Sheet as the number of OD260 units. The amount of oligonucleotide expressed in nanomoles and micrograms is derived from the OD measurement.


Calculate the number of nanomoles present given an OD reading and extinction coefficient: Nanomoles = ( OD260 / ε260) × 106

Example:
1 OD260 unit of primer M13 Forward,
5’-GTA AAA CGA CGG CCA GTG-3’
Molar extinction coefficient (ε260) = 182.800 L / (mole x cm)
Nanomoles = (1.0 / 182.800 ) × 106 = 5.47 nmoles


Convert the amount in nanomoles to micrograms: Micrograms = Molecular Weight × Nanomoles × 10-3

Example
: 1 OD260 unit of primer M13 Forward,
5’-GTA AAA CGA CGG CCA GTG-3’
Molecular Weight = 5558.7
Micrograms = 5558.7 × 5.47 × 10-3 = 30.4 μg



> To quantify your Oligonucleotides, make an aliquot of the resuspended Oligonucleotides to a final volume of 1 ml of dH2O and vortex for a few seconds. Measure the absorbance of this dilution at 260 nm (A260). Use the formula below to calculate the concentration of Oligonucleotides in your stock solution.

This formula is valid for an absorption of A260 ≤1.2.

Concentration in µg/ml =

A260 x dilution factor x Weight per OD of stock solution (in µg/OD)

 

Calculation of the molar extinction coefficient





where ΣNearest Neighbour is the nearest neighbour constant for a pair of bases, ΣIndividual is the constant for an individual base, and n is the length of the oligonucleotide.


Calculation of the molecular weight





For DNA bases:
MW dA = 313.21; MW dC = 289.18; MW dG
= 329.21; MW dT = 304.20; MW dU
= 290.17; MW dI = 314.19

For RNA bases: MW DNA counterpart + 16. When determining the weight of Uracil (rU) start with dU and not dT

For LNA bases: MW dA = 313.21; MW dC = 289.18; MW dG = 329.21; MW dT = 304.20; MW dU = 290.17; MW dI = 314.19

For 2’ O-Methyl bases: MW DNA counterpart + 30.03. When determining the weight of mU start with dU and not dT

For phosphorothioated bases: MW DNA counterpart + 16.06

Mixed bases


Mixed bases (also known as degenerate or wobble bases) follow the IUB codes:

  • 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

Oligonucleotides made using mixed bases result in a final product that is a heterogeneous population of distinct species. MW, Tm and extinction coefficient may be strongly affected by mixed base addition. Rather than reporting the various values for each component, a single value is given.

Chemistries


Chimeric sequences follow the codes:

  • ACGTA = DNA
  • (ACGUA) = RNA
  • [ACGUA] = 2' O-Me RNA
  • {ACGTA} = LNA®
  • A*C(G*U*)A = Phosphorothioates
Number of PCR reactions Quantity of DNA (µg) OD260 Synthesis scale (nmol)
50-500 33 1 10
200-2000 132 4 40
600-6000 396 12 200
1000-10000 660 20 1000

Table 1. Oligonucleotide scale determination for different number of PCR reactions (Final volume: 100 μl, Final concentration of Oligonucleotides: 0.5 μM, Oligonucleotide length: 20 bases).


Number of PCR reactions Quantity of DNA (µg) OD260 Synthesis scale (nmol)
300-600 15 0.5 10
600-1200 30 1 40
2000-4000 90 3 200
4000-8000 200 6 1000

Table 2. Oligonucleotide scale determination for different number of Real-Time qPCR reactions (Final volume: 50 or 25 μl, Final concentration of Oligonucleotides: 100 nM, Oligonucleotide length: 20 bases).


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