Stop and talk with your advisor. qPCR standards need to be double-stranded DNA. Based on the mass & length, your oligo is single-stranded.

why do you even need copy number for?

It is not clear what you are asking about.

1/ If you want to know the copy number (number of molecules) of the oligo in the tube your received:

The amount of the oligo you obtained is 4 nmol. 1 mol is 6.02214076×10^23 copies/molecules (the Avogadro Number). Hence 4 nmol= 2.40886*10^15 copies/molecules of your oligo. Depending on the volume of water/buffer you dissolve it in, you can then get different concentrations.

2/ If you want to quantitate e. g. the amount of a gene in a sample:

You need to perform a control experiment with known amounts of dsDNA as the template to obtain a standard curve. In parallel, you run your sample and by comparing the number of PCR cycles you needed to get a threshold amount of your target gene with the standard curve you calculate the amount of the gene (i.e. the number of molecules containing your gene in your sample).

To determine the copy number of custom oligos for qPCR standards, follow these steps:

1. Calculate the Molecular Weight of the Oligo:

For a double-stranded DNA oligo:

For a single-stranded DNA oligo:

2. Determine the Oligo Mass in Grams:

Convert the oligo concentration from nanomolar (nM) or picomolar (pM) to grams:

3. Calculate the Copy Number:

Use Avogadro’s number to convert the mass to the number of copies:

Simplified Formula:

When the oligo concentration is in ng/µL:

Example:

For a 100 bp double-stranded DNA oligo at 10 ng/µL:

• Molecular Weight = g/mol

• Copy Number = copies/µL

Important Considerations:

• Ensure accurate quantification of the oligo (e.g., using a Nanodrop or Qubit).

• Dilute the oligo appropriately to generate standard curves for qPCR.

• Validate the standard curve’s efficiency (90-110%) and linearity (R² > 0.99).

Would you like help with designing the standard curve or analyzing the qPCR data statistically?