DNA Quantification

Beer-Lambert Law states?

higher concentration of a substance will result in higher absorbance

Formula for concentration of extract

50 ug/mL DNA x OD260 value x DF

Formula for DF

Final volume/aliquot volume

Formula for purity

OD260/OD280

Electrophoresis conditions

100 V for a run time of 30 minutes

Organic compounds and chaotropic salts maximally absorb at

230 nm

During the run, DNA fragments move through a micro- or nanofluidic channel and the sample is separated by electrophoresis

Capillary Electrophoresis

___ reacts with deoxyribose sugars under ___ conditions and forms a ___ complex that can be quantified at ___

Diphenylamine, acidic, blue, 595 nm

Define spectroscopy

Interaction between electromagnetic radiation and matter

Visible light range

370 to 780 nm

UV light range

100 to 400 nm

Method of performing spectroscopy

Spectrophotometry

3 aromatic amino acids

1. Phenylalanine (1 ring)

2. Tyrosine (1 ring)

3. Tryptophan (2 rings)

Formula for Beer-Lambert’s Law

A = log(I₀/I) = εcl

Fundamental relationship of Beer-Lambert’s Law (direct relationship)

Concentration, path length, and molar absorptivity are all directly proportional to the absorbance

Fundamental relationship of Beer-Lambert’s Law (inverse relationship)

Transmittance/transmitted light is inversely proportional to absorbance

Pore size to concentration of gel

Higher concentration of gel ⇒ smaller pores

Concentration of gel for short DNA

higher agarose concentration

Purpose of borate in TBE

Provides ions to carry the charge across the gel

5 components of AGE

1. 0.5x TBE

2. Agarose powder

3. Gel stain/intercalating gel dye

4. DNA loading dye/DNA loading buffer

5. DNA ladder

Components of DNA loading dye (3)

Main solvent is Tris buffer with glycerol and tracking dye

If a lane is heavily saturated, it means?

High concentration of DNA

What do smears in a lane signify after DNA extraction

small fragments or low-quality DNA

Range of purity

1.8–2.0 (high purity) | <1.73 (contaminated)