Gel Electrophoresis Khan Academy

Gel electrophoresis

A lab technique used to separate DNA or proteins based on their size using a gel and an electrical field.

Purpose of gel electrophoresis

To separate biological molecules (DNA or proteins) by size so they can be visualized, compared, or isolated.

"Gel" in gel electrophoresis

The medium through which molecules travel; can be made from substances like agarose or polyacrylamide.

"Electrophoresis"

Movement of charged molecules in an electric field; in this technique, it moves DNA/proteins through a gel.

Electrical field in gel electrophoresis

Created by a cathode (negative, reduction) and an anode (positive, oxidation), connected to a power source.

Cathode

The negatively charged electrode where reduction occurs; placed at one end of the gel apparatus.

Anode

The positively charged electrode where oxidation occurs; placed at the opposite end of the gel.

Power supply

A battery or power source used to create the electrical field across the gel.

Buffer in gel electrophoresis

Ion-containing solution that covers the gel and conducts electricity, allowing current to pass through.

Sample loading

DNA or protein samples are pipetted into wells at one end of the gel.

Loading dye

Colored dye mixed with samples so their movement can be tracked during electrophoresis (colors in diagram: pink, yellow, green).

Tracking dye colors (example)

Pink, yellow, and green shown in diagrams; colors are arbitrary and just help visualize movement.

DNA charge

DNA has a negatively charged backbone due to phosphate groups, causing it to migrate toward the anode (positive end).

Direction of DNA migration

From cathode (negative) to anode (positive).

Early gel pattern (short run)

Bands may not be well separated; e.g., pink sample splits into several bands, yellow has one band, green has two close bands.

Longer run outcome

Bands separate more clearly by size; fragments spread out across the gel according to length.

DNA ladder

A standard mixture of DNA fragments of known sizes, loaded alongside samples to compare and estimate unknown fragment sizes.

Purpose of DNA ladder

To serve as a size reference; allows identification of the base-pair lengths of unknown DNA fragments.

Example ladder sizes

Common example: 400 bp, 200 bp, and 100 bp fragments.

Relationship between fragment size and migration

Smaller DNA fragments migrate faster and farther; larger DNA fragments move slower and less far.

Example: 400 bp fragment

Moves the shortest distance on the gel (large, harder to push).

Example: 100 bp fragment

Moves the farthest on the gel (small, easy to push).

Yellow sample result

Contains a single DNA fragment of 200 base pairs.

Green sample result

Contains two fragments: one 200 bp and one 100 bp.

Practical application of gel electrophoresis

Fragments of desired size can be cut out of the gel and used for cloning, insertion into plasmids/vectors, sequencing, or other molecular biology techniques.

Two common gel types

Agarose and SDS-PAGE (polyacrylamide).

Agarose gel

Gel type with large pores, used for separating large DNA fragments (>50 base pairs).

Agarose pore size

Relatively large; good for separating big pieces of DNA but not very effective for small fragments.

SDS-PAGE gel

Polyacrylamide gel with small pores, used for separating small DNA fragments or proteins.

SDS in SDS-PAGE

Sodium dodecyl sulfate; a detergent that denatures proteins by disrupting non-covalent interactions.

Function of SDS

Removes shape and charge effects from proteins, ensuring separation is strictly based on size.

PAGE in SDS-PAGE

PolyAcrylamide Gel Electrophoresis; refers to the gel matrix material.

Why SDS-PAGE separates by size only

SDS makes proteins uniformly negatively charged, so migration depends only on size, not native charge or shape.

Memory trick for SDS-PAGE

"S for Small, S for SDS" → SDS-PAGE is for small DNA fragments or proteins.

Memory trick for agarose

Agarose is for larger DNA fragments (big pore size).

Comparison of agarose and SDS-PAGE

Agarose = large fragments (>50 bp DNA); SDS-PAGE = small DNA fragments or proteins.

Visualization of bands

Bands represent DNA fragments of different sizes; separation improves with longer electrophoresis time.

Final experimental use

Once fragment sizes are determined, DNA/protein can be sequenced, cloned, or used in further molecular techniques.