Molarity Calculations#
When collecting DNA/RNA from a sample it is important to measure the amount that you have. In biology labs, this is typically done by measuring the absorbance of light at specific wavelengths.
Calculating Concentration#
Nucleic acids absorb light at 260 nanometers. This can be used to weigh the amount of DNA in a sample like so:
\(\text{Concentration (ng/μL)} = A_{260} \times \text{Conversion Factor}\)
With the following standard conversion factors.
Nucleic Acid |
Conversion Factor |
|---|---|
DS-DNA |
50 ng/uL |
SS-DNA |
33 ng/uL |
SS-RNA |
40 ng/uL |
Calulating Molarity#
Oftentimes when performing enzymatic reactions it is important to know the number of molecules per unit volume. This is called the molarity of a sample. By controling the number of DNA molecules added to a sample we can ensure they are enough enzyme molecules to properly cataylze the reaction.
Since DNA fragments can be different lengths, we need to employ a conversion that calculates the weight of each molecule of DNA. This will be specific to each experiment as different protocols will generate DNA fragments of different lengths.
Assume we have a 250 basepair double-stranded piece of DNA and we want to add 25 femptomoles of DNA to our reaction.
We can calculate how much each mole weighs using:
\(250bp×660g/mol=165,000g/mol\)
Using this, we can calculate the number of moles per μL in our sample:
\(\text{Moles/μL} = \frac{\text{Mass/μL}}{\text{Molar Mass}} = \frac{100 \times 10^{-12}}{165,000}\)
\(\text{Moles/μL} = 6.06 \times 10^{-16} \, \text{mol/μL}\)
6.06E-16 moles/μL is a valid answer, however, most protocols use femptomoles as the unit of choice.
\( 1 \text{mol}=10^{15} \text{femptomols} \)
\(\text{Molarity of the DNA sample} = 6.06 \, \text{fmols/μL}\)
Therefore, to add 25 femptomoles of our DNA to a solution we would:
\( \frac{25 μL}{6.06 \text{fmols/μL}} = 4.1 μL\)
In the walkthrough and lab you will employ these techniques to determine the efficiency of different protocols.
Extra Resources#
Below are additional links to review unit conversion.
Khan Academy’s Unit Conversions
Bite Size Bio has a DNA focused example.
New England Biosciences Calculators are a great in-lab resource.