Visualization

Electrophoresis

Movement of charged molecules toward an electrode of the opposite charge. Used to separate charged molecules.

Agarose

A gel forming polysaccharide found in certain types of seaweed.

Purified agarose

Agarose that is in powdered form.

Insoluble agarose

The powder is insoluble in water (or buffer) at room temperature.

Dissolving agarose

Agarose dissolves when boiled.

Polymerization of agarose

As gel cools, it undergoes polymerization, causing the solution to 'gel' into a semi-solid matrix much like 'Jello' only more firm.

Agarose concentration effect

The more agarose is dissolved in the boiling water, the firmer the gel will be.

Casting tray

While the solution is still hot, it is poured into a mold called a 'casting tray'.

Electrophoresis chamber

An electrophoresis chamber and power supply.

Gel casting trays

Composed of UV-transparent plastic.

Sample combs

Around which molten agarose is poured to form sample wells in the gel.

Electrophoresis buffer

Usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).

Loading buffer

Contains something dense (e.g. glycerol) to allow the sample to 'fall' into the sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring of how far the electrophoresis has proceeded.

Ethidium bromide

A fluorescent dye used for staining nucleic acids. Ethidium bromide is a known mutagen and should be handled as a hazardous chemical - wear gloves while handling.

Transilluminator

An ultraviolet lightbox used to visualize ethidium bromide-stained DNA in gels.

DNA fragment migration

Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to the log10 of their molecular weight.

Circular DNA migration

Circular forms of DNA migrate in agarose distinctly differently from linear DNA of the same mass.

Resolution of DNA fragments

Higher concentrations of agarose facilitate separation of small DNA fragments, while low agarose concentrations allow resolution of larger pieces of DNA.

Optimal agarose concentration for large fragments

Larger fragments are much better resolved in the 0.7% gel.

Optimal agarose concentration for small fragments

Small fragments separate best in 1.5% agarose.

Voltage effect on fragment migration

As the voltage applied to a gel is increased, larger fragments migrate proportionally faster than small fragments.

Best resolution voltage

The best resolution of fragments larger than about 2 kb is attained by applying no more than 5 volts per cm to the gel.

Buffer recommendations for electrophoresis

Several different buffers have been recommended for electrophoresis of DNA.

TAE

Tris-acetate-EDTA, a commonly used buffer for duplex DNA.

TBE

Tris-borate-EDTA, another commonly used buffer for duplex DNA.

Ionic strength

A factor that causes DNA fragments to migrate at different rates in TAE and TBE buffers.

Buffers

Substances that establish pH and provide ions to support conductivity.

Agarose gels

Medium where ethidium bromide can be incorporated to visualize DNA fragments.

Concentrated buffer

Using a 10X stock solution may generate enough heat in the gel to melt it.

Binding of ethidium bromide

Alters the mass and rigidity of DNA, affecting its mobility.

Migration of DNA

Essentially nonexistent if water is mistakenly used instead of buffer.

Pulsed field electrophoresis

A technique in which the direction of current flow in the electrophoresis chamber is periodically altered, allowing fractionation of pieces of DNA ranging from 50,000 to 5 million bp.

Alkaline agarose gels

Prepared with and electrophoresed in buffers containing sodium hydroxide, useful for analyzing single-stranded DNA.

Polyacrylamide gels

Restrains larger molecules from migrating as fast as smaller molecules.

Better separation of molecules

Refers to the improved resolution achieved in electrophoresis.

Can visualize smaller fragments

Indicates the capability of certain gels to detect smaller DNA or protein fragments.

Polyacrylamide gels flexibility

Offers greater flexibility and more sharply defined banding than agarose gels.

SDS

Sodium dodecyl sulfate, an anionic detergent that denatures proteins and confers a negative charge.

PAGE

Polyacrylamide gel electrophoresis, separation of proteins under denaturing conditions.

Migration determination

Migration is NOT determined by intrinsic electrical charge of the polypeptide, but by molecular weight.

Buffer Systems

Includes continuous systems with a single separating gel and the same buffer in tanks and gels, and discontinuous systems with different buffers for stacking and resolving gels.

Acrylamide/bisacrylamide monomer stock solution

A solution used in the preparation of polyacrylamide gels.

4 X Running gel buffer

Contains 1.5 M Tris-HCL, pH 8.8.

4 X stacking gel buffer

Contains 500 mM Tris-HCL, pH 6.8.

SDS Stock Solution

A 10% (w/v) solution of sodium dodecyl sulfate.

Ammonium persulfate initiator solution

A 10% (w/v) solution used to initiate polymerization in gel preparation.

5 X Electrode (running) buffer

A concentrated buffer used in electrophoresis.

5 X Non-reducing treatment buffer

A concentrated buffer used for non-reducing conditions in protein analysis.

5 X Reducing treatment buffer

A concentrated buffer used for reducing conditions in protein analysis.

β-mercaptoethanol

A reducing agent commonly used in protein electrophoresis.

Molecular markers

Standards used to determine the size of molecules in gel electrophoresis.

Two-dimensional gel electrophoresis

Begins with 1-D electrophoresis of protein, then separates the analytes by a second property in a direction 90 degrees from the first.

Capillary electrophoresis

The adaptation of traditional gel electrophoresis into a capillary format.

Molecular sieve

Uses polymers in solution to create a medium that allows separation based on size.

Southern Blot

A technique where DNA is cut with restriction enzymes and probed with radioactive DNA.

Northern Blot

A technique where RNA is probed with radioactive DNA or RNA.

Western Blot

A technique used to detect biomolecules.

Protein

Probed with radioactive or enzymatically-tagged antibodies.

Gel electrophoresis

A method for separating molecules based on their size.

Transfer to solid support

The process of moving separated molecules to a solid medium for further analysis.

Blocking

Soaking filters in a solution containing a high concentration of DNA, RNA, or protein to prevent non-specific binding of probes.

Preparing Probe

Creating a labeled copy of a double-stranded DNA fragment for detection.

Hybridization

The process where a probe binds to its target molecule.

Washing

The step to remove unbound probes from the filter.

Detection of probe-target hybrid

Identifying the presence of a specific biomolecule using a labeled probe.

Molecular weight (DNA)

Measured in base-pairs (bp) and commonly in kilobase-pairs (kbp), where 1 kbp = 1000 bp.

Molecular weight (RNA)

Measured in nucleotides (nt) and commonly in kilonucleotides (knt), where 1 knt = 1000 nt.

Molecular weight (Protein)

Measured in Daltons (Da) and commonly in kiloDaltons (kDa), where 1 kDa = 1000 Da.

Blotting

The transfer process of separated molecules to a solid support.

Solid support

Usually a sheet of nitrocellulose paper where biomolecules are transferred.

Blocking solution

A solution containing high concentrations of biomolecules to coat the filter and prevent probe binding.

Labels for probes

Includes radioactivity, autoradiography, fluorescence, and chemiluminescence.

Dot blot

A technique where a mixture containing the molecule to be detected is applied directly on a membrane.

Advantage of Dot blot

Offers significant savings in time since electrophoresis and complex blotting procedures are eliminated.

Disadvantage of Dot blot

Offers no information on the size of the molecule; different sizes appear as a single blot.

Presence confirmation

Dot blot can only confirm the presence or absence of a biomolecule.