Molecular Biology Lab: Mini-Prep and Gel Electrophoresis

Vocabulary practice flashcards covering plasmid DNA extraction (mini-prep), restriction enzyme digestion, and agarose gel electrophoresis from Genetics Lab.

Mini-prep

A small-scale procedure that uses a few milliliters of bacterial cells to lyse the cells and extract plasmid DNA.

Kanamycin

An antibiotic added to LB broth to selectively kill bacterial cells that have not been infected with the plasmid and therefore lack resistance.

E. coli Envelope Barriers

The three structural layers separating E. coli from the outside world: the plasma membrane, the peptidoglycan layer, and the outer membrane.

Lysozyme

An enzyme naturally found in lysosomes that digests peptidoglycan to assist in breaking open bacterial cell walls during a mini-prep.

Triton X-100

A detergent included in STET buffer that dissolves the bacterial plasma and outer membranes.

STET Buffer

A lysis formulation composed of sucrose for osmotic support, Tris to maintain pH, EDTA to chelate divalent cations and block nucleases, and Triton X-100 to dissolve membranes.

EDTA (in STET)

A chelating agent that binds divalent cations in solution to prevent nucleases from degrading DNA.

E. coli Chromosome

The circular bacterial genome containing approximately $4.6\text{ million}$ base pairs and around $3000$ genes, making it roughly $1000\times$ larger than a typical plasmid.

Plasmid

An extrachromosomal DNA molecule typically measuring several thousand base pairs in length and capable of carrying half a dozen to several hundred genes.

Chromosomal vs. Plasmid Separation

The separation of genomic and plasmid DNA achieved by boiling followed by centrifugation, which precipitates chromosomal DNA into a pellet while leaving plasmid DNA in the supernatant.

Hydrophilic Nature of DNA

The water-attracting property of DNA resulting from the polarity and resting negative charge of its sugar-phosphate backbone.

Alcohol Precipitation of DNA

The formation of a stable ionic salt that falls out of solution when alcohol (such as isopropanol or $70\%$ ethanol) is added, as alcohol is less polar than water and drives negative DNA to pair with cations.

Beta-galactosidase

The enzyme responsible for determining bacterial colony color on selection plates; its large portion is encoded on the E. coli chromosome and its small portion is encoded in the cloning vector.

Palindromic DNA Sequence

A double-stranded DNA sequence (such as GAATTC recognized by Eco RI) that reads identically on both complementary strands when reading in the $5'$ to $3'$ direction.

Eco RI

A restriction endonuclease originally derived from E. coli that cleaves at the recognition site GAATTC, exhibiting optimal activity at $37\,^\circ\text{C}$.

DNA Electrophoretic Migration

The movement of DNA molecules toward the positive anode when placed in an electric field, propelled by the net negative charge of the phosphodiester backbone.

Intercalator

A flat, planar compound (such as ethidium bromide or GelGreen) that inserts between adjacent base pairs of DNA, disrupting replication and causing single base-pair frameshift mutations.

DNA Marker (DNA Ladder)

A standardized set of DNA fragments of known molecular weights and lengths run alongside experimental samples on a gel to calibrate and determine band sizes.

Loading Buffer

A pre-run solution containing a dense component (such as sucrose or glycerol) to sink samples into agarose wells and a low-molecular-weight tracking dye (such as bromophenol blue) to visually monitor electrophoresis.

Agarose

A seaweed-derived gelatinous polysaccharide that forms a porous molecular sieve through which charged nucleic acids migrate during gel electrophoresis.