Course Number & Title: Biol 52 Advanced Biotechnology
Units & Course Hours: 4 units, 144 hours
Weekly Hours: 2 lecture, 6 laboratory
Number of Weeks: 18
Repeatability: 0
Credit/Applicability:

Baccalaureate Degree Applicable
AA/AS Degree Applicable
Certificate Applicable

Course Description

The emphasis of this course will be use of laboratory equipment and lab skills and development of an independent project.

When the student has successfully completed this course he/she will be able to:

1. Successfully maintain cell cultures.
2. Be able to isolate and purify chromosomal and plasmid DNA from cells.
3. Be able to set up electrophorectic runs and analyze the results.
4. Successfully transform bacterial cells and prove that transformation occurred.
5. Use restriction enzymes and analyze products.
6. Set up and perform DNA blots.
7. Understand DNA probes, their principles and applications.
8. Perform in vitro PCR and analyze results.
9. Be proficient in GLPs and GMPs.
10. Design, perform, document, evaluate, and defend an independent project in molecular biology.

• DNA Isolation 3 weeks
• Electrophoresis 2 weeks
• Transformations and Transfections 2 weeks
• Restriction Enzymes and Analysis 3 weeks
• Blot and Probe Techniques 3 weeks
• PCR Techniques and Analysis 3 weeks
• Quality Control and Quality Assurance 2 weeks

1. Critical thinking example.
2. Reading example.
3. Content review and prerequisites

The Case of the Bloody Knife

Late one April night, government agents received an anonymous tip that the National Art Museum was about to be robbed of a priceless jewel collection. When they arrived at the museum, they saw they were too late: the jewels were gone. Lying facedown on the floor next to the empty jewel case was the body of a man the chief inspector recognized as the international jewel thief Heinrich Milhouse. Milhouse had been shot in the chest at close range; his clothes were saturated with blood. Underneath the body, the inspectors found a bloody knife.

At the airport the next day, police apprehended Englewood Smink, the murdered thief’s occasional partner in crime. Smink denied all knowledge of the murder and the theft. When asked about the fresh cut on his hand, Smink said that he had had an accident in the kitchen that morning.

Suspicious, the chief inspector ordered DNA tests on the victim, the blood on the victim’s clothes, the blood on the end of the knife found under the victim, and Smink. Police laboratory technicians used the polymerase chain reaction to look at two different chromosome regions that contain a variable number tandem repeat. They used on set of primers for each region. The chromosome regions, primers, and results of the tests are shown in Fig. 27.1.

What is your interpretation of the data? State your reasons. Should Smink be released? Should other tests be performed?

Fig. 27.1 Results of polymerase chain reaction analysis.

Chromosome region 1:

5’-TCCGAGCTGGACGTGCAG…variable number of TAGA repeats…GTTACACGCCTGAGTTACGGT-3’

3’-AGGCTCGACCTGCACGTC…variable number of ATCT repeats…CAATGTGCGGACTCAATGCCA-5’

Primer set 1: 5’- CCGAGCTGGACGTGCAG-3’ + 3’- AATGTGCGGACTCAATG – 5’
 
 

Chromosome region 2:

5’- CGACGCTTAGCATGTCCAG…variable number of CCAGT repeats…CGCTAGTCGACGCCATC - 3’

3’- GCTGCGAATCGTACAGGTC…variable number of GGTCA repeats…GCGATCAGCTGCGGTAG - 5’

Primer set 2: 5’ – GACGCTTACGATGTCC – 3’ + 3’ – GCGATCAGCTGCGGTA – 5’

EXERCISE 1. Gel Electrophoresis of Nucleic Acids.
 

Nucleic acid samples are often subjected to gel electrophoresis to characterize the size and number of different fragments in the sample. In this exercise, DNA samples that have been digested with restriction enzymes and mixed with a tracking dye will be subjected to agarose gel electrophoresis. The electrophoresis buffer, the salt solution that both conducts electric current and controls the pH of the solution during the separation of the DNA fragments, is TRIS/borate/EDTA or TBE, a commonly used buffer system.

When charged molecules are placed in an electric field, the molecules will migrate towards one of the electrodes, depending on the net charge of the molecule. Nucleic acids have an overall negative charge due to the negative charge associated with the phosphate backbone of the molecules, so they will migrate towards the positive electrode. Since the distribution of phosphate is very regular across the length of the nucleic acid molecule, nucleic acids have a constant change/mass ratio and will therefore migrate at the same rate in an electric field.

Separation of nucleic acid molecules of different size or conformation is achieved by adding a support matrix to the electric field and forcing the molecules to migrate through this matrix, typically agarose or polyacrylamide gel. This matrix acts like a sieve that allows small molecules to go faster than large molecules. The result is that molecules separate in the matrix according to their relative size and shape.

This exercise will use gel electrophoresis to examine the fragments present in several DNA samples. The principle goal is to obtain experience in pouring gels, loading DNA samples, and visualizing the DNA bands.

Due to small volumes of sample to be applied to the gel, samples are routinely handled with an manual micropipet device that uses a disposable plastic tip to handle a 10 to 20 m l sample. Several commercial devices are available. A substitute sample loader can be assembled from a disposable 0.5 or 1 ml syringe fitted with a disposable plastic tip (see Appendix).

Materials

EcoRI and HindIII digested bacteriophage lambda DNA samples containing SM Dye (other DNA samples can be added or substituted).

Reaction Stop Mix Dye (SM Dye):

10% glycerol
5% SDS (sodium dodecyl sulphate)
0.025% xylene cyanol FF dye (XC)
0.025% bromphenol-blue WS dye (BPB)

TBE electrophoresis buffer: 
(can be stored as 10X stock)

90 ml TRIS-HCl, pH 8.2
2.5 ml Na₂EDTA
89 ml boric acid

Ethidium bromide (EtBr). For staining gels to visualize DNA, approximately 200 ml of 4 m g/ml ethidium bromide in water in a shallow tray, stored covered and protected from excessive light exposure. EtBr should be treated as a mutagen—a compound capable of causing genetic mutations—and bare skin should not come in contact with the solution. It is both photosensitive and biodegradable and dilute solutions are often disposed of as biohazardous chemical waste.

Protocol

1. Use the molten agarose to pour a minigel as demonstrated or according to instructions for the gel unit in use. Illustrations for assembling a generic gel unit are included in the Appendix. It is important to be certain that all small agarose particles are completely melted before use. Allow the gel to completely harden before removing the well-forming comb. This should take about 15 minutes.
2. Remove the comb from the solidified gel and transfer the gel to a running unit. Submerge the gel in TBE buffer.
3. Use a manual pipettor with a disposable plastic tip to load 10 m l of each sample into one of the wells in the gel. Plasmid and bacteriophage DNA samples should be easy to load, but chromosomal DNA samples may be quite viscous and difficult to load.
4. Once the samples have been loaded, close the unit and apply power to the electrophoresis chamber. Typical minigels run at 100-130 volts (60-160 milliamps), depending on the unit used. During the run, the XC and BPB dyes will resolve into two bands, with the BPB the faster band. Run the gel until this band is near the end of the gel, then turn the current off and remove the gel.
5. Transfer the gel to the staining tray containing the ethidium bromide solution, and stain the gel for 5 minutes. Ethidium bromide is a mutagen, and gloves should be worn or a spatula used to transfer the gel in and out of the ethidium solution.
6. Transfer the stained gel to a destaining tray containing water. Destain the gel for 5 minutes to remove excess ethidium bromide.
7. The stained gel can be visualized with a UV transilluminator or a UV mineral lamp. DO NOT LOOK DIRECTLY AT THE UV LIGHT!! UV light causes skin burns, is a mutagen, and will cause severe headaches from eye damage with direct exposure. Always use a face mask or shield.

The results of a typical gel are shown illustrated below.

If lambda DNA samples digested with different restriction endonucleases are present in the same gel, note that each different restriction enzyme produces a distinct, completely reproducible pattern of DNA fragments. Relative separation of individual fragments is dependent on agarose concentration, electrophoresis conditions, and the quality of the gel preparation.

Many different types of artifact can distort the bands in a gel. Some of the more common gel artifacts are illustrated below.

1. Gel was crooked in gel box and samples migrated off the gel.
2. Agarose was not completely melted before gel was poured. The small specks of high concentration agarose in the gel cause distortion in DNA bands and intensely spots in the gel.
3. The sample well containing the EcoRI digested lambda DNA leaked at the right side, causing loss of sample intensity and blurring of bands.
4. The smearing of the EcoRI digested lambda DNA is caused by loading of too much DNA.

Department: Life Science Date: 5/11/97
Target Course: BIOL 52 Advanced Biotechnology
Prerequisite: BIOL 31

The student should possess the following skills or knowledge:

1. Knowledge of safety regulations, GLPs and federal regulations relating to biotechnology.
2. Knowledge of appropriate use, storage, and disposal of wastes and biohazards.
3. Knowledge of appropriate use, calibration, maintenance and troubleshooting of biotech lab equipment.
4. Knowledge of centrifuges, incubators, automatic pipettors, spectrophotometers, electrophoretic equipment.
5. Knowledge of HPLC, cell counters, and other selected biotechnical equipment.
6. Knowledge of Standard Operating Procedures (SOPs), collection, interpretation and presentation of data.
7. Knowledge of culturing and maintaining bacteria using aseptic technique.
8. Knowledge of preparation of biological solutions to precise concentrations and dilutions.
9. Knowledge of DNA mechanics, gene expression and nucleic acid manipulations.
10. Knowledge of tissue culture using sterile technique.
11. Knowledge of protein separation and analysis.
12. Knowledge of immunological methods of testing.

Number of items with a mean rating of 3 or greater is 12. Percentage of items with a mean rating of 3 or greater is 100%.

Department Recommendation: X Prerequisite

Completed by: Janet Fulks, Janice Toyoshima, Kenward Vaughan, Wendell Wall, Tom Yale

Department: Life Science Date: 5/13/97
Target Course: BIOL 52—Advanced Biotechnology
Prerequisite: Organic Chemistry—CHEM 11, CHEM 16 or CHEM 8/9

The student should possess the following skills or knowledge:

1. An understanding of the importance of carbon’s ability to form strong bonds with itself and other commonly found elements, thus forming the backbone for large, chemically complex molecules required for the existence of life.
2. An understanding of basic valence bond theory, its application to the bonding of atoms to one another, and the consequences that such bonding confers on local and overall molecular structure and reactivity.
3. An understanding of oxidation/reduction processes as well as some primary types of reactions (hydrolyses, dehydrations, condensations, etc.) found in organic substances.
4. An understanding of the structure of functional groups, the reactivity/characteristics they confer upon a molecule, and their role in the chemical manipulation of organic substances.
5. An understanding of the types of inter- and intramolecular interactions which exist in organic substances, their dependence on external factors (e.g., pH and ionic strength), and the consequences all of these have on various physical and chemical characteristics of biochemicals (e.g., protein and DNA structure, and enzymes functioning as chemical catalysts).

Number of items with a mean rating of 3 or greater is 6. Percentage of items with a mean rating of 3 or greater is 100%.

Department Recommendation: X Prerequisite

Completed by: Janet Fulks, Janice Toyoshima, Tom Yale, Wendall Wall, Kenward Vaughan

The SCANS five competencies and three-part foundation skills are incorporated into an integrated (vocational and academic), sequenced program that includes school and work-based learning. To what extent are these competencies being met in this course:

Directions: Circle the number that best describes the degree to which each component is taught. 1 = 0-25% 2 = 26-50% 3 = 51-75% 4 = 76-100% 

SCANS Competencies

Competency 1	Resources: Identifies, Organizes, Plans and Allocates Resources
1      2     3     4	TIME—selects goal relevant activities, ranks them, allocates time, and prepares and follows schedules
1      2     3     4	MONEY—uses or prepares budgets, makes forecasts, keeps records, and makes objectives to meet objectives
1      2     3     4	MATERIAL AND FACILTIES—acquires, stores, allocates and uses materials or space efficiently
1      2     3     4	HUMAN RESOURCES—assesses skills and distributes work accordingly, evaluates performance and provides feedback
Competency 2	Interpersonal: Works with Others
1      2     3     4	PARTICIPATES AS A MEMBER OF A TEAM—contributes to group efforts
1      2     3     4	TEACHES OTHERS NEW SKILLS
1      2     3     4	SERVES CLIENTS/CUSTOMERS—works to satisfy customers’ expectations.
1      2     3     4	EXERCISE LEADERSHIP—communicates ideas to justify position, persuades and convinces others, responsibly challenges existing procedures and policies
1      2     3     4	NEGOTIATES—works towards agreements involving exchange of resources, resolves divergent interests
1      2     3     4	WORKS WITH DIVERSITY—works well with men and women from diverse backgrounds
Competency 3	Information: Acquires And Uses Information
1      2     3     4	ACQUIRES AND EVALUATES INFORMATION
1      2     3     4	ORGANIZES AND MAINTAINS INFORMATION
1      2     3     4	INTERPRETS AND COMMUNICATES INFORMATION
Competency 4	Systems: Understands Complex Inter-Relationships
1      2     3     4	UNDERSTANDS SYSTEMS—knows how social, organizational, and technological systems work and operates efficiently with them
1      2     3     4	MONITORS AND CORRECTS PERFORMANCE—distinguishes trends, predicts impacts on system operations, diagnoses deviations in systems performance and corrects malfunctions
1      2     3     4	IMPROVES OR DESIGNS SYSTEMS—suggests modifications to existing systems and develops new or alternative systems to improve performance
Competency 5	Technologies: Works With A Variety of Technologies
1      2     3     4	SELECTS TECHNOLOGY—chooses procedures, tools or equipment including computers and related technology
1      2     3     4	MAINTAINS AND TROUBLESHOOTS EQUIPMENT—prevents, identifies, or solves problems with equipment, including computers and other technologies
Foundation Skills
Skill 1	Basic Skills: Reads, Writes, Performs Arithmetic and Mathematical Operations, Listens and Speaks
1      2     3     4	READING—locates, understands and interprets written information in prose and in documents such as manuals, graphs and schedules
1      2     3     4	WRITING—communicates thoughts, ideas, information, and messages in writing; and creates documents such as letters, directions, manuals, reports, graphs and flow charts
1      2     3     4	ARITHMETIC/MATHEMATICS—performs basic computations and approaches practical problems by choosing appropriately from a variety of mathematical techniques
1      2     3     4	LISTENING—receives, attends to, interprets, and responds to verbal messages and other cues
1      2     3     4	SPEAKING—organizes ideas and communicates orally
Skill 2	Thinking Skills: Thinks Creatively, Makes Decisions, Solves Problems, Visualizes, Knows How to Learn And Reason
1      2     3     4	CREATIVE THINKING—generates new ideas
1      2     3     4	DECISION MAKING—specifies goals and constraints, generates alternatives, considers risks, evaluates and chooses best alternative
1      2     3     4	PROBLEM SOLVING—recognizes problems and devises and implements plan of action
1      2     3     4	SEEING THINGS IN THE MIND’S EYE—organizes and processes symbols, pictures, graphs, objects and other information
1      2     3     4	KNOWING HOW TO LEARN—uses efficient learning techniques to acquire and apply new knowledge and skills
1      2     3     4	REASONING—discovers a rule or principle underlying the relationship between two or more objects and applies it in solving a problem
Skill 3	Personal Qualities: Displays Responsibility, Self-Esteem, Sociability, Self Management, and Integrity and Honesty
1      2     3     4	RESPONSIBILITY—exerts a high level of effort and perseveres toward goal attainment
1      2     3     4	SELF ESTEEM—believes in own self-worth and maintains a positive view of self
1      2     3     4	SOCIABILITY—assesses self accurately, sets personal goals, monitors progress and exhibits self control
1      2     3     4	INTEGRITY/HONESTY—chooses ethical courses of action
Knowledge of "All Aspects of the Industry"
Means strong experience in, and understanding of, all aspects of the industry the students are preparing to enter.
1      2     3     4	Employers and school personnel jointly design learning outcomes and participate in curriculum development and approval
	The instructional program (vocational and academic, school and work-based) include strong experience in, and knowledge, of the following aspects of the industry on which the instructional program is based:
1      2     3     4	Planning
1      2     3     4	Management
1      2     3     4	Finances
1      2     3     4	Technical and Production Skills
1      2     3     4	Underlying Principles of Technology
1      2     3     4	Health and Safety
1      2     3     4	Staff development efforts enhance necessary skills and appropriate attitudes for faculty, counselors, administrators, workplace instructors and supervisors
1      2     3     4	Work-based activity explicitly reinforces academic and technical lessons
1      2     3     4	Students are engaged in real, productive work
1      2     3     4	Other
1      2     3     4	Other
1      2     3     4	Other

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