Lab 9: DNA Gel Electrophoresis and SDS Page

Gel Electrophoresis

a procedure used to separate biomolecules such as DNA, RNA, or proteins using a gel matrix made of agarose and polyacrylamide

Separation

depends on the properties of the gel and the molecules being separated

Gel Matrix

contains pores of a uniform size through which molecules can pass through

Pore Size

depends on gel material, agarose makes larger pores than polyacrylamide

Gel Concentration

determines the size of the pores; the higher concentration of the gel, the smaller the pores will be; larger molecules pass less easily through small pores

Nucleic Acids: DNA/RNA

very large molecules that are generally separated using agarose gel

Polyacrylamide

used to separate proteins that are generally much smaller than DNA and RNA

Molecule Size

most important factor for migration through the gel matrix, larger molecules migrate slower than smaller molecules

Supercoiled DNA

native structure and most stable form of DNA

Coiled DNA

when coiled, DNA size is very small and compact, allowing it to move faster through the gel matrix compared to the linear fragment formed after cutting with a restriction enzyme

Nicked DNA

generated when one of the DNA strands is cut/hydrolyzed in crude DNA isolation and acts like a hook,holding DNA to the gel

DNA Sample

mixed with a DNA loading buffer and loaded in the wells

DNA Loading Buffer

a concentrate containing a tracking dye(s) and glycerol

Blue Tracking Dye

has a very low molecular weight and runs ahead of the DNA fragments and tracks progress of the colorless DNA through the gel

Glycerol

increases the density of the sample and makes it easier for it to fall into the wells and prevents it from floating away

DNA Marker

contains a mixture of DNA fragments of known sizes is used to determine the size of DNA in the samples

Digested (Cut) and Non-Digested (Uncut) DNA Controls

used to compare and analyze the gel observations

Sharper Bands

occur when DNA is loaded in the smallest volume possible

Electric Current

molecules require electrical current to move/migrate through the gel pores; the higher the voltage, the faster the DNA moves

High Voltage

heats the gel and causes it to gel, denature the DNA, and decrease the resolution (separation) of the DNA fragments

Charge

affects migration of molecules through an electrical field; when current is applied, cations move towards cathode, anions move towards anode (moves toward opposite charge, positive to negative)

Molecules Charge and Size

molecules with the same size but different charges differ in migration, molecules that differ only in size must have a similar charge

Why Water is Not Used

not used for preparing gels or used as a running solution because it is not a good conductor of electricity

Running Buffer (TAE Buffer pH 8)

contains electrolytes that are used to ensure that electricity conducts through the gel

DNA and RNA

contains ionizable phosphate groups and are negatively charged at pH 8

TAE Buffer (Tris-Acetate-EDTA)

preferred to other buffers such as TBE (Tris-Borate-EDTA) because it provides better separation of fragments > 4 kb; gel is covered with 2-3 mm with buffer

Too Much TAE Buffer

results in heating the gel, decreases DNA migration, and distorts DNA bands

SYBR Safe DNA Gel Stain

a highly sensitive stain for visualization of nucleotide strands in agarose or acrylamide gels

SYBR Safe

binds with double stranded DNA to form a complex that fluoresces under UV light, can also bind to single stranded RNA

Size of DNA Fragments

estimated by comparing their sizes with the DNA marker

Lambda DNA

digested with Hind III enzymes is often used as a DNA marker for sizing and to approximate quantification of DNA

Low Concentration of DNA

smaller bands are invisible

DNA Shapes

double helix but can fold and coil itself into more complex shapes

DNA Shapes: Cut

when cut upon digestion with enzymes, it unfolds and gets longer

DNA of Most Organisms

negatively supercoiled

Nicked DNA: DNA Shapes

DNA may become nicked during extraction process and create hooks in the circular DNA