Plasmid Purification & Quantification LAB

Flashcards covering key concepts from the Plasmid Purification & Quantification lecture notes.

Selectable Marker

A gene on a plasmid that allows only the cells containing that plasmid to survive and grow under specific conditions.

Classes of Selectable Markers

Two major classes: Antibiotic resistance genes (e.g., KanR, AmpR) and Nutritional markers.

pJA0013 Characteristics

Plasmid number: pJA0013, Selectable marker: Kanamycin resistance (KANR), Insertion: sfGFP, Host: DH5α, Size: 4716 bp.

pJA0017 Characteristics

Plasmid number: pJA0017, Selectable marker: Ampicillin resistance (AMPR), Insertion: mRFP, Host: DH5α, Size: 5806 bp.

MCS (Multiple Cloning Site)

A short engineered segment on a plasmid containing many restriction enzyme recognition sites for DNA fragment insertion.

Agarose

A high–molecular-weight polysaccharide from seaweed, forming a gel matrix for DNA electrophoresis.

SDS in Agarose Electrophoresis

SDS is not needed for DNA as it already has a negative charge; it is used for protein denaturation in SDS-PAGE.

Structural Forms of Plasmids

Three forms: Nicked/open circular, Linearized, Supercoiled.

Migration of Plasmid Forms

Supercoiled migrates fastest, Linearized is moderate, Nicked/open circular migrates slowest.

Absorbance of Nucleic Acids

Nucleic acids absorb most strongly at 260 nm.

Absorption at 260 nm

The conjugated double bonds in purine and pyrimidine rings of nucleotides absorb strongly at this wavelength.

ABS260 and DNA Quantity

Absorbance at 260 nm is directly proportional to DNA concentration.

Absorption at 280 nm

Aromatic amino acids in proteins (tryptophan and tyrosine) absorb at 280 nm.

ABS260/280 Ratio

Indicates purity: High ratio → purer nucleic acid; Low ratio → higher protein contamination.

Ideal ABS260/280 Ratios

DNA: ~1.8; RNA: ~2.0.

Low ABS260/280 Ratio Implication

A ratio lower than 1.8 indicates protein contamination.