Applied Molecular Biology: Cloning, Expression, and Purification of Taq DNA Polymerase

Comprehensive practice flashcards covering terminology, reagents, and procedures for cloning, expression, and purification of Taq DNA polymerase.

Miniprep

A small-scale purification procedure used to extract plasmid DNA from an overnight bacterial culture using alkaline lysis and a silica-based spin column matrix.

P1 buffer

A cell suspension buffer containing RNase that is used to resuspend pelleted bacterial cells prior to cell lysis.

P2 buffer

A cell lysis buffer containing NaOH and SDS that breaks open bacterial cells and denatures chromosomal DNA and proteins.

N3 buffer

A neutralisation buffer containing guanidine hydrochloride, potassium acetate, and acetic acid that precipitates genomic DNA, protein, and cell debris while keeping plasmid DNA soluble.

SybrGold

A fluorescent nucleic acid gel stain that binds to DNA and RNA and fluoresces when exposed to blue or UV light.

NanoDrop spectrophotometer

A micro-spectrophotometer used to measure nucleic acid concentration in $\text{ng/}\,\mu\text{L}$ using the double-stranded DNA conversion factor of $1\,\text{OD}_{260}\text{ unit} = 50\,\mu\text{g DNA/mL}$.

EcoRV

A restriction endonuclease that recognizes a specific hexanucleotide sequence and cleaves DNA to generate blunt ends.

Buffer QG

A gel-dissolving buffer containing guanidine thiocyanate, chaotropic salts, and a pH indicator that turns yellow at $\text{pH} \le 7.5$ to ensure optimal DNA binding to the silica column.

Buffer PE

An ethanol-containing wash buffer used to remove residual salts and impurities from silica spin columns without eluting the bound DNA.

TE buffer

A buffer consisting of Tris and EDTA at $\text{pH } 7.5$ used to elute and store DNA while protecting it from nuclease degradation through divalent cation chelation.

Sticky-end ligation

The enzymatic joining of DNA fragments that possess complementary single-stranded overhangs, such as those generated by a BamHI restriction digest.

Blunt-end ligation

The enzymatic joining of double-stranded DNA fragments that lack single-stranded overhangs and terminate with fully base-paired ends, such as those produced by an EcoRV digest.

pETBlue™-1

A $3476\,\text{bp}$ bacterial plasmid vector containing a T7lac promoter, lacZα sequence, ampicillin resistance marker ($\text{AmpR}$), and multiple restriction sites designed for high-level inducible protein expression in E. coli.

Alkaline phosphatase

An enzyme used to dephosphorylate the $5'$ termini of linearized vector DNA to prevent vector self-ligation (re-ligation) in the absence of an insert.

T4 DNA ligase

An enzyme that catalyses the formation of a phosphodiester bond between adjacent $5'\text{-phosphate}$ and $3'\text{-hydroxyl}$ termini in double-stranded DNA using ATP as an energy cofactor.

DTT (Dithiothreitol)

A reducing agent included in enzymatic reaction and storage buffers to maintain free sulfhydryl groups and preserve enzyme activity.

Competent cells

Bacterial cells treated with cold $\text{CaCl}_2$ solutions to alter membrane permeability, enabling them to take up exogenous plasmid DNA during heat shock.

Heat-shock transformation

A bacterial transformation method in which chilled competent cells mixed with plasmid DNA are rapidly incubated at $42^\circ\text{C}$ for exactly $90\,\text{seconds}$ followed by rapid chilling on ice.

SOC medium

A nutrient-rich recovery broth added to transformed bacterial cells immediately after heat shock to allow phenotypic expression before plating onto antibiotic selection plates.

Carbenicillin

A β-lactam antibiotic incorporated into growth agar to select for bacterial cells carrying plasmids containing the β-lactamase resistance gene ($\text{AmpR}$).

X-GAL

A chromogenic substrate that is cleaved by active β-galactosidase to produce an insoluble blue compound, allowing visual screening of colonies.

IPTG

A non-hydrolysable galactose analogue that induces transcription driven by the lac or T7lac promoter by binding and inactivating the Lac repressor protein.

lacZ gene

A bacterial gene encoding β-galactosidase; insertion of foreign DNA into its multiple cloning site disrupts enzyme production, resulting in white colonies during blue/white screening.

Lysozyme

An enzyme present in Buffer A that breaks down the peptidoglycan cell wall of bacteria to facilitate gentle cell lysis.

Buffer A

A cell lysis suspension buffer containing $50\,\text{mM}\text{ Tris-HCl pH } 7.9$, $50\,\text{mM}\text{ glucose}$, and $1\,\text{mM}\text{ EDTA}$, supplemented with $2\,\text{mg/mL}\text{ lysozyme}$.

Buffer B

A cell lysis extraction buffer containing $10\,\text{mM}\text{ Tris-HCl pH } 7.9$, $50\,\text{mM}\text{ KCl}$, $1\,\text{mM}\text{ EDTA}$, and the detergents $0.5\%\text{ Tween 20}$ and $0.5\%\text{ NP-40}$.

Thermostability-based purification

A protein purification strategy exploiting the extreme heat resistance of Taq polymerase by heating lysates to $75^\circ\text{C}$ for $30\,\text{minutes}$, causing host E. coli proteins to denature and precipitate while leaving Taq soluble.

Sample 1 (Taq purification)

An aliquot ($100\,\mu\text{L}$) of the crude lysed cell suspension kept on ice to serve as an unheated total protein reference control for subsequent SDS-PAGE analysis.

Sample 2a (Taq purification)

An aliquot ($100\,\mu\text{L}$) of clear supernatant collected after heat incubation of Sample 2 at $75^\circ\text{C}$ and centrifugation, kept on ice to evaluate purification efficiency via SDS-PAGE.

Taq Storage Buffer

A stabilizing buffer containing $50\,\text{mM}\text{ Tris-HCl pH } 7.9$, $100\,\text{mM}\text{ NaCl}$, $1\,\text{mM}\text{ DTT}$, $1\%\text{ Triton X-100}$, and $50\%\text{ glycerol}$ mixed with purified Taq enzyme for long-term storage.

Sodium Dodecyl Sulfate (SDS)

An anionic detergent that denatures proteins and binds to polypeptides at a constant ratio, conferring a uniform negative charge proportional to polypeptide length.

SDS-PAGE

A polyacrylamide gel electrophoresis technique used to separate denatured proteins strictly by their molecular mass independently of native conformation.

Coomassie Blue

An organic dye used to stain polyacrylamide gels post-electrophoresis by non-specifically binding to proteins, allowing visualization of distinct bands after destaining in water.

Stain-free SDS-PAGE

A rapid protein visualization technique that utilizes trihalo compounds cast into the gel to covalently label tryptophan residues upon UV/blue light activation without needing post-run liquid staining.

1Kb Plus DNA Ladder

A molecular weight DNA standard consisting of fragments ranging from $100\,\text{bp}$ to $15{,}000\,\text{bp}$, featuring an intense reference band at $1{,}500\,\text{bp}$.