## Scale determination

The table below allows you to determine the right synthesis scale based on the final oligo concentration (in 100 µL),
the desired amount of PCR reactions and the minimum quantity of oligo you have to order.

#### Final Oligo Concentration

50 nM 150 nM 300 nM 600 nM 900 nM
Average number of Reactions (total volume 100μL) Minimum quantity to order*
100 30 15 8 6 0,5 nmol
1000 300 165 80 55 5 nmol
5000 1650 830 415 275 25 nmol
10 000 3300 1660 830 555 50 nmol
100 000 33 300 16 660 8330 5555 500 nmol

Please select in the minimum guaranteed yield table the adequate synthesis scale corresponding to the minimum desired amount.

## Quantitation

#### How to calculate the oligo concentration from absorbance at 260 nm?

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)

#### How Optical Density (O.D.) at 260 nm is linked to oligo quantity?

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 measure the OD260 for each Oligonucleotide and calculate their amount in nanomoles and micrograms.

We report these values on the Product Data Sheet.

#### How to convert OD260 into nanomoles?

The following equation shows the relation between the oligo amount in nanomoles and the OD 260 value

Nanomoles = (OD260/ ε260) x 106
ε260 is the extinction coefficient at 260 nm

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</p

#### How to convert nanomoles into micrograms? nmol to mg

Using the following equation, it is quite simple to calculate the amount in microgram from the nanomole value and the Molecular weight of the oligonucleotide.

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

#### How to calculate the molar extinction coefficient?

ε260 = 2 .(Σ1n-1 ε Nearest Neighbour) - Σ2n-1 ε Individual + Σ1n ε Modifications

Where ε Nearest Neighbour is the nearest neighbour constant for a pair of bases, ε Individual is the constant for an individual base, and n is the length of the oligonucleotide.

#### How to calculate the molecular weight of my oligonucleotide?

Anhydrous MW (g.mol-1) = Σindividual base MW + Σindividual Mods MW - 63.98 + 2.016

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 DNA counterpart + 16 (+42 for dC)
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