QUALITY ASSESSMENT

Quality assessment

Process of evaluating the concentration and purity of DNA or RNA to assess its suitability for further laboratory procedure

Spectrophotometry

Method that measure the amount of light absorbed by a sample of specific wavelength

Nanodrop

Microvolume UV-Vis spectrophotometer that used to measure the concentration and purity of DNA and RNA using a very small sample volume of 1-2 microliter

Beer-lamber law

Absorbance is directly proportional to the sample concentration

260 nm

Nucleic acid: DNA and RNA show their main absorbance

280 nm

Proteins: primarily connected with the absorbance of aromatic amino acids, especially tryptophan and tyrosine

230 nm

Contaminants: absorbance may indicate contamints such as phenol, salts and other organic compounds

320-340 nm

Baseline correction: can be used to assess background absorbance caused by turbidity or other sample effect

Absorbance

In beer-lambert law, a means?

Molar extinction coefficient

In beer-lambert law, e means?

Concentration

In beer-lambert law, c means?

Path length

In beer-lambert law, l means?

Xenon flash lamp

High intensity UV/Vis light source

Pedestal and arm

Uses surface tension to hold 1-2 microliter sample droplets without cuvettes

Optical fibers

Transmits light through the sample along defined short pathlengths

CCD spectrometer

Detects and measures light intenstiy after passing through the sample

Embedded software

Automatically processes absorbance readings and calculates nucleic acid concentrations and purity ratios

Absorbance

Amount of light absorbed by the sample at a specific wavelength

Path length

Distance that light travels through the sample

260/280 ratio

Ratio used to assess protein contamination

1.8

Expected value of pure dna

2.0

Expected value of pure dna

<1.6-1.7

Expected value of protein contamination or phenol residual

260-230 ratio

Ratio used to assess whether there are chemical or organic contaminatns

2.0-2.2

Expected value of pure nucleic acid

<1.8

Expected value of presence of chemical salts, guadinine, or carbohydrates

Protein

PCR reaction fails because DNA polymerase is inhibited.

What contaminant is most likely?

Phenol

Residual extraction solvent remains after purification.

What contaminant is most likely?

Rna contamination

Dna sample contains unwanted RNA molecules.

What contaminant is most likely?

Dna degradation

DNA appears fragmented after improper storage.

What contaminant is most likely?

Ethanol

Washing step was incomplete, leaving alcohol residue.

What contaminant is most likely?

Fluorometry

Measures nucleic acid concentration using fluorescent dyes

Electrophoresis

Evauates dna or rna fragment integrity

Spectrophotometry

Measures nucleic acid purity using ultraviolet light

Fluorometry

Highly sensitive for low concentration samples

Electrophoresis

Visualizes DNA bands on agarose gel

1. DNA/RNA extraction

2. Measure quantity

3. Assess quantity

4. Interprets results

5. Suitable for PCR/sequencing

Quality control process