Cytoskeleton and Molecular Biology Lab Techniques

Vocabulary terms and definitions related to PCR methodology, primer design specifications, and principles of agarose/polyacrylamide gel electrophoresis.

Polymerase Chain Reaction (PCR)

A method of duplicating DNA chains involving repeated heating and cooling of a sample under laboratory conditions to amplify a specific DNA fragment.

Kary Mullis

The scientist from the Californian company Cetus who developed the PCR technique in 1983 and was awarded the Nobel Prize in 1993.

PCR Template Quantity (Human)

The recommended maximum amount of human genomic DNA matrix for a standard PCR reaction is $500\,ng$.

Annealing Temperature ($T_a$)

The attachment temperature of primers, which is usually $\sim 5$ to $10^{\circ}C$ lower than the melting temperature ($T_m$) and calculated as $T_a = T_m - 5^{\circ}C$.

Primers (Starters)

Short DNA oligonucleotides, typically $19-25\,nt$ in length, that are complementary to the matrix fragments at both ends of the gene of interest.

Forward Primer

A type of primer whose sequence must be the same as the DNA sequence being duplicated.

Reverse Primer

A type of primer whose sequence must be the reverse complement of the DNA sequence being duplicated.

Melting Point Formula ($T_m$)

For primers no longer than 25 nucleotides, the formula used is $T_m = 4(G + C) + 2(A + T)$.

Electrophoresis

A method of separating DNA, RNA, and proteins in a suitable electrolyte under the influence of an applied voltage based on size, charge, and conformation.

Agarose

A polysaccharide derived from kelp that forms a spatial network with water molecules, used for the separation of large molecules like nucleic acids.

Agarose Phase Transitions

Agarose dissolves in water at approximately $85-100^{\circ}C$ and solidifies at approximately $32-45^{\circ}C$.

Ethidium Bromide (EtBr)

An intercalating dye that penetrates the hydrophobic region between adjacent nucleotides, increasing its fluorescence intensity under UV light.

Polyacrylamide Gels

Carriers with finer cross-linking than agarose, widely used for the electrophoretic separation of proteins and small sections of DNA.

Ammonium Persulfate (APS)

A substance used to provide chemical activation to initiate the radical polymerization reaction of acrylamide.

TEMED

($N, N, N', N'\text{-tetramethylethylenediamine}$); used as a catalyst in polymerization to accelerate the release of free radicals.

Bis-acrylamide

A compound present in the polyacrylamide gel mixture that acts as a cross-linker to stabilize polymer chains and form a porous carrier.

Vertical System

An electrophoretic spatial orientation most commonly used for protein separation using polyacrylamide gels.

Horizontal System

An electrophoretic spatial orientation preferred for nucleic acid separation using agarose gels.

Sodium Dodecyl Sulphate (SDS)

A detergent that unfolds proteins by breaking stabilizing bonds and masks their original charge with a negative charge for weight-based separation.

$$\beta\text{-mercaptoethanol}$$

A compound used to destroy disulfide bridges in proteins, enabling better electrophoretic separation and a linear molecular structure.

Glycerol

A loading agent that allows the test sample to be held and precisely positioned in the well of the concentration gel.

Bromophenol Blue

A negatively charged dye that migrates with the sample on the gel, allowing the user to follow the progress of the migration.

TBE Buffer

A mixture of Tris-base, boric acid, and EDTA used as a continuous buffer system for nucleic acid electrophoresis.

EDTA

A component in electrophoresis buffers whose function is to minimize the aggregation and degradation of nucleic acids.

Electrophoretic Marker

A parallel separated standard containing molecules of known molecular weight used to estimate the size of the fragments in the analyzed sample.