Molecular Biology Practical Manual Flashcards

Vocabulary flashcards covering safety guidelines, reagents, principles, and procedures for genomic DNA and RNA extraction, spectrophotometry, gel electrophoresis, and UV mutagenesis protocols.

Cetyl trimethyl ammonium bromide (CTAB)

A powerful cationic detergent used in plant DNA extraction that solubilizes plant cell walls and lipid membranes of internal organelles while denaturing proteins.

Polyvinylpyrrolidone (PVP)

A chemical used in plant DNA extraction alongside CTAB to minimize the interference and co-precipitation of secondary metabolites like polyphenols with DNA.

Beta-mercaptoethanol

A reducing agent included in extraction buffers that denatures proteins by cleaving disulfide bonds between cysteine residues and helps remove tannins and polyphenols.

EDTA (Ethylene Diamine Tetraacetic Acid)

A chelating agent that binds divalent cations such as $Mg^{2+}$, inhibiting DNase activity and helping disintegrate cell walls.

Tris Buffer

A buffering agent commonly used at pH 8.0 in DNA extraction protocols to maintain a stable pH environment for nucleic acids.

Sodium Chloride (NaCl)

A monovalent salt that supplies metal ions to neutralize the negative phosphate charges on DNA, facilitating DNA aggregation and precipitation.

Phenol-Chloroform Extraction

A classical liquid-liquid extraction technique using phenol, chloroform, and isoamyl alcohol to partition denatured proteins into an organic layer or interphase while leaving nucleic acids in the aqueous phase.

Isoamyl Alcohol

A reagent added to the phenol-chloroform mixture (25:24:1) that aids in reducing foaming and removing remaining protein and carbohydrate contaminants.

Proteinase K

An enzyme added during cell lysis to digest cellular proteins and ensure complete removal of protein contaminants from nucleic acid extracts.

Ethidium Bromide (EtBr)

An intercalating agent that binds between DNA base pairs and emits orange-red fluorescence under ultraviolet (UV) light.

Gel Loading Dye

A reagent containing tracking dyes and density agents (such as glycerol) that increases sample density so DNA sinks into agarose gel wells.

Guanidinium Thiocyanate

A strong chaotropic agent used in RNA extraction to denature proteins, disrupt cell structure, and rapidly inactivate endogenous ribonucleases (RNases).

Cyclobutane Pyrimidine Dimers (CPDs)

The most common DNA lesions induced by UV light (254 nm), formed primarily between adjacent thymine bases (T-T) on a DNA strand.

6-4 Pyrimidine-Pyrimidone Photoproducts

UV-induced DNA lesions that occur at lower frequency than cyclobutane pyrimidine dimers and are repaired more efficiently by cellular mechanisms.

Photoreactivation

An error-free, light-dependent DNA repair pathway in E. coli that reverses UV-induced pyrimidine dimer damage.

rpoB Gene (rif)

A genetic locus located at approximately 90 centisomes on the E. coli K-12 chromosome where mutations confer resistance to the antibiotic rifampicin.

lacZ Locus

A genetic marker located at approximately 8 centisomes on the E. coli chromosome; mutants lacking this functional gene ($\text{lacZ}^-$) cannot utilize lactose.

Freeze-Thaw Method

A convenient physical cell disruption procedure using alternating temperatures (dry ice-ethanol bath and $95^\text{o}\text{C}$ water bath) to lyse yeast cell walls for genomic DNA extraction.

SDS (Sodium Dodecyl Sulfate)

An anionic detergent that solubilizes membrane lipids and denatures membrane-bound proteins during cell lysis.

RNase A

An endoribonuclease enzyme used during DNA purification to rapidly digest contaminating RNA molecules.