6b lab

6b lab - Dhaval Rana Ap Bio January 4, 2010 Lab 6b: Cutting...

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Dhaval Rana Ap Bio January 4, 2010 Lab 6b: Cutting DNA Using Restriction Enzymes and Gel Electrophoresis Abstract: The objective of this lab is to separate three samples of DNA from the bacteriophage lambda using gel electrophoresis. One sample of DNA is cut with the restriction endonuclease EcoRI, one cut with the restriction endonuclease HindIII, and one uncut control. The hypothesis is that if shorter fragments of DNA migrate further than longer fragments, then the DNA digested with EcoRI will migrate further than the DNA digested with HindIII. In this lab, the DNA samples are loaded into wells of an agarose gel and electrophoresed. An electrical field applied across the gel causes the DNA fragments in the samples to move from their origins (sample wells) through the gel matrix toward the positive electrode. The restriction patterns are made visible by staining with the Carolina BLUTM, which binds to DNA. Smaller DNA fragments migrate faster during electrophoresis. The characteristic number and pattern of bands produced by each restriction enzyme is, in effect, a “DNA fingerprint.” Introduction: Bacteria have enzymes that cut, or "digest," the DNA of foreign organisms and thereby protect the cells from invaders such as viruses. Several hundred of these enzymes, known as restriction enzymes, have now been isolated. Each is able to recognize and cut at a specific DNA sequence, known as a recognition sequence. The discovery of restriction enzymes made genetic engineering possible because researchers could use them to cut DNA into fragments that could be analyzed and used in a variety of procedures. In this part of the laboratory, we will use gel electrophoresis to separate samples of DNA that have been digested by restriction enzymes. Then we will compare fragments of unknown size to fragments of a known size to calculate the unknown fragment sizes. Like all enzymes, restriction enzymes are highly specific. They cut DNA only within very precise recognition sequences. All of these recognition sites are symmetrical, or what is called "palindromic." This means that the recognition sequence on one DNA strand reads in the opposite direction on the complementary strand. We begin by mixing DNA with one or more restriction enzymes in a small plastic
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This document was uploaded on 10/15/2010.

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6b lab - Dhaval Rana Ap Bio January 4, 2010 Lab 6b: Cutting...

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