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Objectives

Students should be able to:

  1. Build a model of the DNA molecule and identify the various components.

  2. Extract and isolate the DNA of onion cells or beef liver cells.

  3. Model transcription and translation of from a DNA sequence to a peptide chain.

  4. Use restriction enzymes to digest DNA and then separate the fragments using gel electrophoresis.

  5. Transform E. coli bacteria by inserting a plasmid for antibiotic resistance.

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Introduction

This set of laboratory exercises will acquaint you with molecular genetics and biotechnology. You will be modeling and manipulating DNA so you may initially understand its structure and chemical / electrical properties. You will also alter genetically alter a bacterium by inserting a plasmid (a circular strand of DNA containing specific genetic information) into selected bacteria.

You will be using various types of equipment, models and materials in order to carry out the lab activities for this exercise. The exercise will require three laboratory periods. You are to turn in the Pre-Lab Questions and the Contributions Sheet prior to lab. Make sure you have done all of the Pre-Lab Activities and have downloaded and printed out any materials, and have received from your instructor the handouts required for lab.

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DNA / RNA

DNA (deoxyribonucleic acid) is one component of the "genetic material". It is composed of four different nucleotide units. A nucleotide is composed of a nitrogen base, (either adenine, thymine, guanine and cytosine), a deoxyribose sugar (C5 or pentose), and a phosphate group. The four nucleotides are named for their nitrogen base, hence they are an adenine nucleotide (A), a thymine nucleotide (T), a guanine nucleotide (G) and a cytosine nucleotide (C). These nucleotides monomers are arranged in particular sequences to form the larger double helix of the DNA polymer. There are several types of DNA found in living organisms.

RNA (ribonucleic acid) is also a polymer built of nucleotides. It is composed of four different nucleotides. Each of the RNA nucleotides is formed from the chemical combination of a nitrogen base, just as in DNA, except in RNA, the nitrogen base thymine is replaced with the nitrogen base uracil. The sugar is ribose instead of deoxyribose. The phosphate group remains the same as in DNA. The polymer is also different because it is a single helix and is very much shorter than DNA.

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Transcription / Translation

The sequence of nucleotides in the DNA molecule can code for the sequence of amino acids in a protein. You will use modeling to help in your understanding of the central dogma of molecular genetics, that of passing the "code" from DNA to RNA to the "building" of a protein. The DNA "code" is transcribed into complementary mRNA (messenger RNA), which is translated into specific amino acids using tRNA (transfer RNA). This process of protein synthesis occurs in the nucleus of the cell (transcription) and then moves to the cytoplasm (translation).

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Restriction Enzymes

Restriction enzymes will be used to fragment a sample of DNA (lambda DNA). Restriction enzymes are found naturally in bacteria and are useful in preventing a bacterium from being colonized by viruses  (bacteriophages, DNA surrounded by a protein sheath). They "attack" the bacteriophage by cutting it into fragments thus the virus is no longer capable of colonizing the bacterium. The restriction enzyme works by cutting the DNA at a specific nucleotide sequence. There are many different restriction enzymes, each cut at a different sequence. One of the restriction enzymes that you will use is EcoRI. EcoRI stands for:

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Escherichia coli = the genus and species of bacterium.
 

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Strain R = the particular strain of E. coli bacteria from which the restriction enzyme was isolated.
 

bullet I (one) means that it is the first restriction enzyme isolated from E. coli, strain R.

EcoRI "finds" and cuts at the DNA sequence:

    GAATTC   (5'---3')  
   CTTAAG    (3'---5') 
           

The above represents a strand of complementary DNA where the letters represent the nucleotides.

EcoRI cuts between the G and the A nucleotide on both strands of the DNA. These cuts produce what is termed a "sticky end". Other restriction enzymes work in a similar manner.

Once DNA is cut it produces DNA fragments of varying length. These fragments can be separated from one another by using a technique called gel electrophoresis.

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Gel Electrophoresis

Gel electrophoresis is a technique that is widely applied in biotechnology. It is used to separate organic molecules, or fragments of molecules, which have an electrical charge on them (usually negative). These organic molecules can be proteins, DNA, RNA, etc. The process is carried out in a chamber that contains a buffer solution (usually TBE). The buffer solution conducts an electrical current, which produces an electrical field in the chamber. The chamber also contains a gel tray which contains a slab of agarose gel that has "wells" for solutions that contain the organic molecules that are to be separated from each other. When the chamber is electrified the negatively charged molecules migrate through the gel and are separated out in the gel. The rate of migration in the gel depends on the molecule's affinity for the current, it size and its shape. The migration of the organic molecules is toward the positive electrode. A picture of the gel electrophoresis equipment can be obtained during Pre-Lab Activity #3 or by clicking here.

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Colony Transformation

Transformation is the process by which a plasmid is incorporated into a bacterium. This process occurs naturally in bacteria. Once biologists understood how the process worked and were able to duplicate it under controlled conditions in the laboratory a whole new area of biotechnology was established. Plasmids are circular extrachromosomal pieces of DNA that carry genetic information separate from the chromosomal DNA of the bacterium. Some plasmids can replicate autonomously and others only when the bacterial chromosome replicates. The plasmids that you will be working with are called R plasmids which carry genes for antibiotic resistance. The specific plasmid for this lab is one that contains a gene (Ampr) that confers resistance to the antibiotic, ampicillin. You will be using the bacterium Escherichia coli (E. coli) for this exercise and will, through a series of processes, transform nonresistant bacteria into resistant ones by making the nonresistant bacteria "competent" so they will absorb the plasmid, pAMP.

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Pre-lab Activities

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Pre-Lab Activity #1 - DNA Extraction

Download and printout the following supplemental laboratory exercise. Please read it before coming to lab:

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Extraction of DNA from Onion Cells

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Pre-Lab Activity #2 - Colony Transformation Information

Download and print out the following:

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Inserting a DNA sample into a plasmid

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Handout on Colony Transformation from the instructor. Please read this material before coming to lab.

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Pre-Lab Activity #3 - Restriction Enzyme and Gel Electrophoresis Information

Download and print out the following:

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Gel Box & Tray Information

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Logarithmic Graph Paper

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Handout on Restriction Enzymes and Gel Electrophoresis from the instructor. Please read this material before coming to lab.

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Pre-Lab Activity #4 - Research

1. Using the resources available in the LRC (Access Science) find and printout the biographies of:

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James Watson

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Francis Crick

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Rosalind Franklin

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Maurice Wilkins

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Linus Pauling

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Frederick Griffith

Read these biographies and list their contributions to the study of DNA. List their contributions using the sheet found by clicking here

2. Watson and Crick's Original Paper - Nature, 1953

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Pre-lab Questions (html version)    Pre-Lab Questions (Word version)

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The Laboratory Activities and Data Collection

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Lab Activity #1 - DNA Modeling

Materials:

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DNA Modeling  Kit


Procedures:

  1. Follow the procedures in the manual that comes with the DNA Modeling Kit.

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Lab Activity #2 - DNA Extraction

Materials:

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Extraction of DNA from Onion Cells

Procedures:

  1. Follow the procedures as outlined in the Extraction of DNA from Onion Cells download.

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Lab Activity #3 - Modeling Transcription / Translation

Materials:

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Handout from the instructor

Procedures:

  1. Follow the procedures as outlined in the handout.

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Lab Activity #4 - Colony Transformation

Materials:

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Handout from the instructor

Procedures:

  1. Follow the procedures as outlined in the handout

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Lab Activity #5 - Restriction Enzyme Fragmentation of DNA and Gel Electrophoresis of DNA Fragments

Materials:

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Handout from the instructor

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Logarithmic Graph Paper

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Discussion Questions     Html format    Word format

Procedures:

  1. Follow the procedures as outlined in the handout.

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Post-lab Activity and Data Analysis

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Results and Analysis:

Below are listed the activities you performed in lab and the results and analysis materials that are to be given to the instructor.

  1. DNA Modeling and DNA Extraction - A brief summary of your results.

  2. Modeling Transcription / Translation -

  3. Restriction Enzyme Fragmentation of DNA and Gel Electrophoresis of DNA Fragments - Discussion questions (answered); logarithmic graph; pAMP sequence map; restriction map data for fragment lengths; and a copy of your gel picture.

  4. Colony Transformation - Results and Discussion questions (in handout) answered

  5. Post-Lab Discussion Questions - Answered

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Post-Lab Questions (html version)    Post-Lab Questions (Word version)

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