Gel Electrophoresis

What is gel electrophoresis?

A technique used to separate molecules, such as nucleic acids or proteins, based on their size, shape, and electrical charge

What are the steps of gel electrophoresis?

1. DNA is extracted from cells (must be a sufficient amount, if not PCR is used)

2. Restriction enzymes cut the DNA at specific nucleotide sequences, producing DNA fragments of different lengths (sizes)

3. DNA samples are mixed with loaded dye and micropipette into wells at one end of an Agarose gel. A DNA ladder (size markers) is also usually loaded into one well for comparison

4. When a current is run through the gel and the buffer it sits in, the DNA fragments move through the gel from the negative electrode (top) to the positive electrode (bottom).

5. The dye mixed prior which fluoresces under UV light, forming bands consisting of the separated fragments. The pattern of this band can be photographed and analysed.

• sugar phosphate group / backbone on DNA molecules is negatively charged, thus opposite charges attract, and it moves towards the positive electrode

What happens to large molecules / DNA fragments?

Move less easily through the pores, thus slower through the gel and are found near the top (negative electrode) of the gel

What happens to small molecules / DNA fragments?

Move more easily through the pores, thus faster through the gel and are found near the bottom (positive electrode) of the gel

What is the purpose of markers?

Markers (which have set, known fragment sizes) are also run in one lane to compare to the unknown fragments to determine their size

Uses for gel electrophoresis

• DNA Profiling

• Crime Scene Analysis

• Paternity Testing

• Evolutionary Analysis

• Species Comparison