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Gel Electrophoresis and
Probes (Southern Blotting)
Group A,
321
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.
321
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