FLUOROMETRY AND NUCLEIC ACID QUANTIFICATION

Fluorometry

Analytical technique used to measure the intensity of fluorescent light emitted by a substance after it has absorbed light of a specific wavelength

Fluoremeter

Used to detect emitted fluorescence

Excitation

Short wavelength, high energy light hits the molecule;

Electrons jump from ground state to an excited state

Vibrational relaxation

Molecule loses a small fraction of energy non-radiatively

Emission

Electrons drop back to ground state, releasing a new photon;

Emitted light generally has a longer wavelength than the excitation light

Fluorometry

Used to determine the concentration of dna and rna

Nucleic acid quantification

Process of determining the amount or concentration of DNA or RNA present in a sample

Fluorometry

Measures the light emitted by molecules after they absorb light of a specific wavelength

Absorbance

Directly measure the amount of a specific wavelength that is absorbed by a sample

Electrophoresis

Determines DNA integrity

Electrophoresis

Estimates DNA fragment size

Spectrophotometry

Measures DNA purity

Spectrophotometry

Measures A260/A280 ratio

Fluorometry

Provides highly sensitive DNA quantification

Fluorometry

Preferred before NGS library preparation

1. Assess DNA integrity

2. Measures DNA purity

3. Measure DNA concentration accurately

4. Perform PCR

Dna extraction procedure

Electrophoresis

What technique should be used to determine whether the DNA is degraded?

Spectrophotometry

Which technique should be used to evaluate DNA purity?

Fluorometry

Which technique should be used to obtain the most accurate DNA concentration before NGS?