BSC 2010L Lab 6-3: Restriction Enzyme Digestion & Core Analytical Instruments

Vocabulary practice flashcards covering restriction digestion, core analytical instrumentation, spectrophotometry, and gel electrophoresis preparation for BSC 2010L Lab 6-3.

Restriction Enzymes

Enzymes found naturally in bacteria that recognize and cut DNA at specific base pair sequences to protect against viral infections by cleaving viral DNA into smaller fragments.

Digestion

The enzymatic process of cleaving DNA into smaller fragments with specific end sequences using restriction enzymes.

Sticky Ends

Unique single-stranded overhang sequences generated in a DNA strand where a restriction enzyme cuts, regardless of the DNA source.

EcoR1

Recognizes the specific palindromic DNA sequence 5'-GAATTC-3' and cleaves it between the guanine and adenine bases to generate a staggered 5' sticky-end overhang.

Pst1

Recognizes the specific palindromic DNA sequence 5'-CTGCAG-3' and cleaves it between the adenine and guanine bases to generate a staggered 3' sticky-end overhang.

Gene Cloning

A molecular biology procedure involving the ligation of a DNA fragment to a cloning vector that has been digested with the same restriction enzyme.

Reaction Buffer H

A buffer solution added to the restriction digestion mixture specifically to optimize enzyme activity.

$$\text{ddH}_2\text{O}$$

Added to the restriction digestion mixture to equate the end reaction volume to $20\,\mu\text{L}$ for perfect enzyme dilution.

Unit of Restriction Enzyme

The specific amount of restriction enzyme required to cut $1\,\mu\text{g}$ of DNA in $1\text{ hour}$.

Consequence of Too Little DNA

An experimental issue where insufficient DNA concentration prevents the digestion fragments from being visualized on an agarose gel.

Consequence of Too Much DNA

An experimental issue that increases the monetary cost of restriction enzyme and requires additional incubation time to complete digestion.

Nanodrop Spectrophotometer

An analytical instrument used to determine the concentration and purity of samples containing biomolecules like DNA, RNA, or proteins.

HPLC (High Performance Liquid Chromatography)

An analytical laboratory instrument used to analyze or purify biomolecules.

Thermocycler

An instrument used to amplify specific segments of DNA via the polymerase chain reaction (PCR).

DNA Sequencer

An instrument used to determine the precise order of the four nitrogenous bases (adenine, guanine, cytosine, and thymine) in a DNA sample.

Absorbance at $260\,\text{nm}$

The specific ultraviolet wavelength at which the nitrogenous bases of DNA and RNA absorb light.

Absorbance at $280\,\text{nm}$

The ultraviolet wavelength at which protein contaminants in a nucleic acid preparation absorb light.

Absorbance at $230\,\text{nm}$

The spectrophotometric wavelength where absorbance indicates contamination by organic compounds, such as salts or ethanol (EtOH).

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

A ratio of absorbance values used to assess DNA sample purity; a value of approximately $1.8$ is generally considered pure DNA.

Nanogram ($\text{ng}$)

A unit of mass equal to $1 \times 10^{-9}\text{ g}$ (one-billionth of a gram), commonly used to express DNA concentration as $\text{ng}/\mu\text{L}$.

Gel Loading Buffer

A solution composed of three main ingredients (EDTA, a density agent, and tracking dyes) that prepares DNA samples for gel electrophoresis.

EDTA

An ingredient in gel loading buffer that acts as a preservative by inhibiting nuclease activity.

Ficoll / Glycerol

Components of gel loading buffer that increase sample density, ensuring the DNA sample sinks to the bottom of the well.

Bromophenol Blue

A tracking dye present in gel loading buffer used to visually monitor the migration of samples through an agarose gel. Faster and smaller than the other blue dye.

Xylene Cyanol

A tracking dye used in gel loading buffer that helps visualize the movement of DNA samples during electrophoresis. Larger and slower than the other blue dye.

Green Fluorescent Protein (GFP)

A reporter protein engineered into plasmids (such as pCellFree_G03) and organisms to visualize and study gene and protein expression.

Lab 6-1

The laboratory exercise focusing on the isolation of eukaryotic DNA from strawberry tissue.

Lab 6-2

The laboratory exercise focusing on the isolation of bacterial plasmid DNA from E. coli, both with and without foreign DNA inserts.

Lab 6-4

The subsequent laboratory exercise designed to separate and visualize digested DNA fragments on an agarose gel by electrophoresis.

Recombinant Bacterium

A bacterial host cell transformed with a recombinant plasmid carrying a foreign target gene, such as the human insulin-producing gene.