Protocol - DNA or RNA concentration from A260nm

Note: Please refer to the attached file (see below) for the formulas, as the formula formatting can be lost/misaligned on websites.

Purpose: To estimate the concentration (nM or uM) of a DNA or RNA solution by determining the absorbance of the solution at 260 nm (A_260nm) and using the Beer-Lambert Law.

Background:

Nucleic acid bases absorb 260 nm light.

According to the Beer-Lambert (or Beer’s) Law, absorbance is proportional to concentration: A = ebc.

A = absorbance (For nucleic acids, use absorbance at 260 nm.)

e = molar extinction coefficient, which is a constant specific for the material tested. This describes how strongly the material absorbs light.

b = pathlength, which is typically normalized to 1 cm.

c = concentration

In essence, we’re using the Beer-Lambert Law, but we’ll set-up the equations just a bit different than the standard A = ebc equation. Understand each step of the calculation.

Questions:

What’s the “molar extinction coefficient”?

Why do we include the length of the oligonucleotide in the calculation?

Can we determine the concentration of an unpurified PCR reaction? How? What things should we consider?

Protocol:

1. Determine the absorbance at 260 nm (A_260nm) of the DNA or RNA sample. Follow the nanodrop instructions, which include properly blanking the instrument.

2. Multiple the absorbance by the constant (37, 40, or 50) for the material to find the “ng/uL” concentration …

a. A₂₆₀ * X = “ng/uL” concentration

i. X = 37 for ssDNA

ii. X = 40 for RNA

iii. X = 50 for dsDNA

3. To find the “nM” concentration, multiple the “ng/uL” concentration by 10⁶. Divide this number by the product of the constant multiplied by the length of the oligonucleotide. That is – perform this calculation

a. (“ng/uL” concentration * 10⁶)

--------------------------------------- = “nM” concentration

[ Y * (length in bases)]

b. Where Y = …

i. Y = 330 g/mol for ssDNA (average base/basepair molecular weights)

ii. Y = 345 g/mol for RNA

iii. Y = 660 g/mol for dsDNA

4. ALTERNATIVELY, instead of finding the “nM” concentration, you may find the “uM” concentration using this calculation …

a. X ng/uL

--------------------------------------- = “uM” concentration = “pmol/uL” concentration

[Y * (length in bases)]

b. Where Y = …

i. Y = 0.330 ng/(pmol * nt) for ssDNA

ii. Y = 0.345 ng/(pmol* nt) for RNA

iii. Y = 0.660 ng/(pmol * bp) for dsDNA

Example:

I used the Nanodrop Spectrophotometer to measure the A_260nm of my primer (40 nt oligonucleotide) solution. What is the uM concentration of the solution?