BIOC 463A Lecture 3: Characterizing an Unknown Plasmid by Restriction Enzyme Mapping

Vocabulary flashcards covering plasmid features, alkaline lysis purification buffers, spectrophotometric purity ratios, restriction enzymes, and agarose gel electrophoresis from BIOC 463A Lecture 3.

Plasmid

A small, circular piece of double-stranded DNA ($dsDNA$) capable of self-replication, stable maintenance, and cloning or carrying foreign DNA.

Ampicillin

An antibiotic that binds to and inhibits enzymes involved in the synthesis of the bacterial cell wall, thereby blocking cell replication.

$\beta$-lactamase

An enzyme encoded by the ampicillin resistance gene ($amp^R$) that is secreted into the periplasmic space of bacteria to hydrolyze the $\beta$-lactam ring of ampicillin.

Type II Restriction Endonucleases

Enzymes that recognize and cut DNA at specific, usually palindromic sequences, producing either blunt ends or $5'$ or $3'$ overhanging sticky ends.

Endonuclease Cut Sites Diagram

A visual guide illustrating recognition sequences and cleavage positions for restriction endonucleases generating blunt ends (HpaI) or staggered overhanging ends (EcoRI, HindIII, PstI).

Buffer P1 (Resuspension Buffer)

A miniprep buffer used to resuspend the cell pellet; contains EDTA, which disrupts lipopolysaccharides (LPS) and chelates divalent cations to inhibit DNase activity, as well as RNase A.

Buffer P2 (Lysis Buffer)

An alkaline lysis solution containing SDS to disrupt lipid structures and denature proteins, and NaOH to raise the pH, which disrupts hydrogen bonding between base pairs and denatures $dsDNA$ into $ssDNA$.

Buffer N3 (Neutralization Buffer)

An acidic potassium acetate solution that neutralizes pH, precipitating genomic DNA, proteins, and cell debris into salt-detergent complexes (white flocculent) while allowing plasmid DNA to renature and remain in solution.

Nitrogenous Bases Structure Diagram

A chemical structural reference showing single-ring pyrimidines (Uracil, Thymine, Cytosine) and double-ring purines (Adenine, Guanine), whose aromatic rings absorb UV light at $\lambda = 260\,\text{nm}$.

$$1\,\text{A}_{260}\text{ Unit of dsDNA}$$

A standard concentration equivalence where an absorbance reading of $1.0$ at $\lambda = 260\,\text{nm}$ corresponds to $50\,\mu\text{g DNA/mL}$.

$$\text{A}_{260}/\text{A}_{280}\text{ Ratio}$$

A absorbance ratio used to assess nucleic acid purity; pure DNA has a ratio of approximately $1.8$ (RNA is approximately $2.0$), whereas lower values indicate protein contamination.

$$\text{A}_{260}/\text{A}_{230}\text{ Ratio}$$

A purity ratio with an expected range of $2.0$ to $2.2$; lower values indicate contamination by carbohydrates, residual phenol, or guanidine, while higher values suggest pedestal contamination or incorrect blanking.

SYBR Safe

A stain used in agarose gel electrophoresis to visualize DNA and RNA bands under blue or UV light.

Plasmid Gel Migration Conformations

The relative mobility of plasmid DNA forms in agarose gel electrophoresis, where supercoiled DNA migrates fastest, linear DNA migrates at an intermediate rate, and nicked/relaxed circular DNA migrates slowest.