Gel Electrophoresis and Probes (Southern Blotting) Group A,

Gel Electrophoresis and

Probes (Southern Blotting)

Group A,

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Before beginning Gel Electrophoresis, test tubes containing identical DNA fragments must be acquired.

Test Tubes

Restriction Enzyme 1

Restriction Enzyme 2

Restriction Enzymes 1 & 2

Restriction enzymes will be introduced in order to cleave the DNA into fragments of different sizes.

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Enzyme 1 is introduced to the first tube, cleaving DNA into fragments A and B.

A B C D EA D

Enzyme 2 is introduced to the next tube, cleaving DNA into fragments C and D.

Enzymes 1 & 2 are introduced to the final tube, cleaving DNA into fragments A, E and D.

1

2

1&2

Size Stds.

23,000 bp – 560 bp

Agarose Gel

A slab of agarose gel has been manufactured with reservoirs to hold the samples of the DNA fragments cleaved by the different restriction enzymes.

Buffer Solution

1

2

1&2

Size Stds.

23,000 bp – 560 bp

Agarose Gel

The gel is placed in a buffer solution to manage pH balance. pH plays a significant role in electrophoresis.

1

2

1&2

Size Stds.

23,000 bp – 560 bp

Agarose GelBuffer Solution

When an electrical charge is applied, the negatively charged DNA fragments flow towards the positive charge. DNA with a lesser amount of base pairs flows faster than those of greater base pairs. Base pair sizes of the samples are determined by a control group of size standards (fragments of known measurement) in one of the reservoirs.

Basic Solution

1

2

1&2

Size Stds.

The basic solution denatures the DNA into single strands

Agarose Gel

Salt Solution

1

2

1&2

Size Stds.

Nylon Filter

Salt Solution

1

2

1&2

Size Stds.

Paper Towels

Paper Towels

Nylon Filter

Agarose Gel

Solution with Radioactive Probes

Solution with Radioactive Probes

Solution with Radioactive Probes

1

2

1&2

Size Stds.

23,000 bp – 560 bp

Agarose GelBuffer Solution

Gel Electrophoresis

A B C D EA D