Synthesis scales and typical yields
For an unmodified 20mer with equal base composition (DNA and RNA):
|Scale||Typical yield Mol. Biol. grade||Typical yield after HPLC|
|0.01 µmol||2-3 nmol||1-2 nmol|
|0.05 µmol||10-15 nmol||5-8 nmol|
|0.20 µmol||30-50 nmol||25-40 nmol|
|1.00 µmol||120-200 nmol||100-150 nmol|
|10.00 µmol||1,000-1,500 nmol||600-1,000 nmol|
|15.00 µmol||2,000-3,500 nmol||1,500-3,000 nmol|
For scales available click here:
What's the difference between scale and yield?
Every oligonucleotide is synthesized in a series of steps and has different properties based on sequence, length and modifications. Thus, the exact yield of a given synthesis reaction using a specific amount of starting material is extremely hard to predict.
Consequently, oligos are ordered by synthesis scale, which refers to the amount of starting material for the first base of the synthesis. The yield is the final amount of produced oligo which is delivered to the customer.
During synthesis and purification there are many factors that affect the final yield of an oligonucleotide. The quality of the synthesis itself plays a crucial role for the oligo yield. The coupling efficiency of a synthesis never occurs with 100% efficiency, but already a very small decrease in coupling efficiency can impact the yield severely. Since every new coupling reaction depends on the efficiency of the one that has happened before, the loss of final yield increases with the length of the oligo.
The synthesis quality can also be influenced by the oligo sequence, such as GC content, as well as by the quality of the chemicals and reagents used. Modified nucleotides and dye labels often have decreased coupling efficiency and thereby negatively impact the yield.
A large amount of oligo material may be lost during purification. This also depends on the amount of failure sequences and contaminants which have to be removed, underlining the importance of an extremely high synthesis quality. The higher the requested purity is, the lower the recovery rate of the final product will be.
An oligonucleotide with secondary structure also complicates the purification and therefore can cause a significant loss of material.
At IBA, we make sure that well-trained staff, good maintenance of synthesizers and the use of high quality reagents set the basis for constantly good yields. See the following table to compare typical yields for different purification grades.