Gel Electrophoresis and Probes (Southern Blotting)

Gel Electrophoresis and Probes (Southern Blotting) Group A,

Before beginning Gel Electrophoresis, test tubes containing identical DNA fragments must be acquired. Restriction enzymes will be introduced in order to cleave the DNA into fragments of different sizes. 1 2 3 Test Tubes Restriction Enzyme 1 Restriction Enzyme 2 Restriction Enzymes 1 & 2

Enzyme 1 is introduced to the first tube, cleaving DNA into fragments A and B. 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 3 C D A E D A B

A slab of agarose gel has been manufactured with reservoirs to hold the samples of the DNA fragments cleaved by the different restriction enzymes. 23,000 bp – 560 bp Size Stds. 1&2 2 1 Agarose Gel

The gel is placed in a buffer solution to manage pH balance. pH plays a significant role in electrophoresis. 23,000 bp – 560 bp Size Stds. 1&2 2 1 Agarose Gel Buffer 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. 23,000 bp – 560 bp Size Stds. 1&2 2 1 Agarose Gel Buffer Solution

The basic solution denatures the DNA into single strands Size Stds. 1&2 2 1 Agarose Gel Basic Solution

Nylon Filter Size Stds. 1&2 2 1 Salt Solution

Paper Towels Size Stds. 1&2 2 1 Salt Solution

Paper Towels Nylon Filter Agarose Gel

Solution with Radioactive Probes

23,000 bp – 560 bp Size Stds. 1&2 2 1 Agarose Gel Buffer Solution

Gel Electrophoresis C D A E D A B

Gel Electrophoresis and Probes (Southern Blotting)