Introduction to Biotechnology

Austin Community College, Biotechnology Department

BIOL 1414

Introduction to Biotechnology

Laboratory Manual

Fall 2011

Linnea Fletcher, Evelyn Goss, Patricia Phelps, Angela Wheeler, and Shelley O`Grady

ISBN: BIOL1414F2011

BIOL1414 Lab Manual Fall 2011

1

BIOL1414 Lab Manual Fall 2011

2

Table of Contents

LAB UNIT 1: INTRODUCTION TO BIOL1414 LAB ................................................................. 5 Lab Unit 1-A: Documentation: The Lab Notebook & Lab Report ........................................... 11 Lab Unit 1-B: Biotechnology Laboratory Security & Safety.................................................... 17 Lab Unit 1-C: Lab Equipment & Reagent Orientation.............................................................. 25 Lab Unit 1-D: Math Skills for the Laboratory........................................................................... 31

LAB UNIT 2: CURRENT GOOD MANUFACTURING PRACTICES (cGMP) ....................... 39 LAB UNIT 3: BASIC TOOLS IN THE BIOTECHNOLOGY LABORATORY........................ 85

Lab Unit 3-A: Using a Micropipette.......................................................................................... 91 Lab Unit 3-B: Calibrating Lab Instruments............................................................................... 97 LAB UNIT 4: PREPARING SOLUTIONS ............................................................................... 103 LAB UNIT 5: TOTAL RNA ISOLATION FROM ALFALFA SPROUTS .............................. 115 Lab Unit 5-A: Total RNA Isolation......................................................................................... 121 Lab Unit 5-B: Denaturing Gel Electrophoresis of Total RNA ................................................ 123 LAB UNIT 6: TRANSFORMATION OF E. coli WITH A RECOMBINANT PLASMID ...... 127 LAB UNIT 7: PLASMID ISOLATION ..................................................................................... 135 LAB UNIT 8: RESTRICTION ENZYME MAPPING .............................................................. 141 LAB UNIT 9: GREEN FLUORESCENT PROTEIN PURIFICATION.................................... 151 LAB UNIT 10: SDS-PAGE OF PURIFIED GFP ...................................................................... 159 LAB UNIT 11: PCR-based VNTR Human DNA Typing .......................................................... 169 Lab Unit 11-A: Isolating DNA & PCR of VNTR Loci........................................................... 171 Lab Unit 11-B: Analysis of VNTR loci PCR Fragments Using Agarose Gel Electrophoresis175 LAB UNIT 12: BIOREMEDIATION ........................................................................................ 179 LAB UNIT 13 ? DNA FINGERPRINTING BY SOUTHERN BLOT ANALYSIS ................. 185 Lab Unit 13-A: Electrophoresis & Transfer DNA .................................................................. 187 Lab Unit 13-B: Colorimetric Detection of DNA ..................................................................... 191 LAB UNIT 14 ? INDIRECT ELISA FOR HIV-1 DETECTION .............................................. 195 LAB UNIT 15 ? MINING BIOLOGICAL DATABASES ON THE INTERNET .................... 203 Lab Unit 15-A: Hemoglobin Bioinformatics........................................................................... 207 Lab Unit 15-B: Bioinformatics Translation Exercise............................................................. 219

BIOL1414 Lab Manual Fall 2011

3

Lab Unit 15-C: Finding Mutant Sequences ............................................................................. 227 APPENDIX................................................................................................................................. 229

Appendix A: Safety Training Sheet........................................................................................ 231 Appendix B: Sample Pre-Lab.................................................................................................. 237 Appendix C: Forms ................................................................................................................. 239 Appendix D: Graphing Data................................................................................................... 245 Appendix E: Summary of Good Laboratory Practices ........................................................... 249 Appendix F: Agarose Gel Electrophoresis with SYBR Green ............................................... 253

BIOL1414 Lab Manual Fall 2011

4

LAB UNIT 1: INTRODUCTION TO BIOL1414 LAB

Welcome to your first course in biotechnology! This course will emphasize its laboratory component to reflect the importance of your training in biotechnology skills. Keep in mind as you work your way through this manual the specific purposes in each exercise. They will prepare you for your first job in a biotechnology laboratory, so keep a careful record of your experience. If you carefully document and archive your work, this information will be easy for you to access later and your experiences will be more valuable in your later work.

The objectives of the lab portion of the course are to: o Develop the basic laboratory techniques of a biotechnology or bioscience lab o Supplement and enrich the lecture portion of the course, which deals predominantly with biotechnology techniques o Develop critical thinking skills in the students o Encourage teamwork and accountability among the students o Practice accuracy in calculations and in writing scientifically o Develop multitasking skills o Encourage students to take charge of their learning o Learn the responsibilities associated with working in a company

Students are expected to behave professionally at all times. Lab notebooks will be maintained and graded in the lab. The lecture will provide background and relevant information about the solutions, prep, procedure and related techniques.

Required Texts/Materials Textbooks: Seidman & Moore, Basic Laboratory Methods for Biotechnology: Textbook &

Laboratory Reference, 2nd edition. 2009. Prentice Hall. ISBN: 0321570146 Lab Manual: BIOL1414 Introduction to Biotechnology Laboratory Manual One copy of the lab

manual will be supplied. Supplies: 3 Ring Binder (Lab notebook), Scientific Calculator with statistics and linear regression

capability, Sharpie labeling pens (fine tip). Lab Safety: Safety glasses with a rating of Z87 and close-toed shoes

Before you can begin working in an ACC laboratory, you must first 1. View the ACC Science Safety video. 2. Tour the laboratory with your laboratory instructor to locate emergency equipment and

procedures. 3. Sign a safety contract, by which you agree to comply with safety regulations.

We hope that you enjoy your experience in this introductory course. Following is a discussion of biotechnology, and a description of some of the activities that you will be doing in this course.

BIOL1414 Lab Manual Fall 2011

5

What is biotechnology? Strictly speaking, biotechnology is the use of a living organism for one`s own benefit. By this definition, biotechnology would date back to the very beginnings of civilization, when humankind first learned to cultivate crops and domesticate animals in a system of agriculture. When one thinks of modern biotechnology, however, gene splicing and recombinant organisms take center stage. Biotechnology was revolutionized when scientists first learned how to isolate and clone genes, allowing for genetic engineering.

Today, the biotechnology industry has grown and expanded to affect us on a day-to-day basis.

Below is an excerpt from that discusses the ever expanding applications of Biotechnology in healthcare, agriculture and energy ().

Biotechnology: Healing, Fueling, and Feeding the World At its simplest, biotechnology is technology based on biology - biotechnology harnesses cellular and biomolecular processes to develop technologies and products that help improve our lives and the health of our planet. We have used the biological processes of microorganisms for more than 6,000 years to make useful food products, such as bread and cheese, and to preserve dairy products.

Modern biotechnology provides breakthrough products and technologies to combat debilitating and rare diseases, reduce our environmental footprint, feed the hungry, use less and cleaner energy, and have safer, cleaner and more efficient industrial manufacturing processes. Currently, there are more than 250 biotechnology health care products and vaccines available to patients, many for previously untreatable diseases. More than 13.3 million farmers around the world use agricultural biotechnology to increase yields, prevent damage from insects and pests and reduce farming's impact on the environment. And more than 50 biorefineries are being built across North America to test and refine technologies to produce biofuels and chemicals from renewable biomass, which can help reduce greenhouse gas emissions. Recent advances in biotechnology are helping us prepare for and meet society`s most pressing challenges. Here's how:

Heal the World Biotech is helping to heal the world by harnessing nature's own toolbox and using our own genetic makeup to heal and guide lines of research by:

Reducing rates of infectious disease; Saving millions of children's lives; Changing the odds of serious, life-threatening conditions affecting millions around the

world; Tailoring treatments to individuals to minimize health risks and side effects; Creating more precise tools for disease detection; and Combating serious illnesses and everyday threats confronting the developing world.

BIOL1414 Lab Manual Fall 2011

6

Fuel the World Biotech uses biological processes such as fermentation and harnesses biocatalysts such as enzymes, yeast, and other microbes to become microscopic manufacturing plants. Biotech is helping to fuel the world by:

Streamlining the steps in chemical manufacturing processes by 80% or more; Lowering the temperature for cleaning clothes and potentially saving $4.1 billion

annually; Improving manufacturing process efficiency to save 50% or more on operating costs; Reducing use of and reliance on petrochemicals; Using biofuels to cut greenhouse gas emissions by 52% or more; Decreasing water usage and waste generation; and Tapping into the full potential of traditional biomass waste products.

Feed the World Biotech improves crop insect resistance, enhances crop herbicide tolerance and facilitates the use of more environmentally sustainable farming practices. Biotech is helping to feed the world by:

Generating higher crop yields with fewer inputs; Lowering volumes of agricultural chemicals required by crops-limiting the run-off of

these products into the environment; Using biotech crops that need fewer applications of pesticides and that allow farmers to

reduce tilling farmland; Developing crops with enhanced nutrition profiles that solve vitamin and nutrient

deficiencies; Producing foods free of allergens and toxins such as mycotoxin; and Improving food and crop oil content to help improve cardiovascular health. Source: Healing, Fueling, Feeding: How Biotechnology is Enriching Your Life

Biotechnology Industry in Austin, Texas The biotechnology industry has also been steadily growing in the Austin area. Today, Austin`s bioscience community encompasses approximately 100 companies that employ more than 7000 people in the areas of research, diagnostics, pharmaceuticals and medical devices. (Ref: ). Some of these companies include Abbott Spine, Agilent, Asuragen, Bioo Scientific, Life Technologies, Luminex, Rules Based Medicine, Viagen, and Zimmer Biologics to name a few! Austin is also a major contributor to academic research in the biological sciences, both at the University of Texas and the University of Texas/M.D. Anderson Cancer Research Center in nearby Bastrop, Texas.

Watch this video to learn more about the exciting and emerging biotech industry in Austin:

To learn more about careers in biotechnology:

BIOL1414 Lab Manual Fall 2011

7

Biotechnology Techniques and Skills Included in This Course The State of Texas has adopted the Washington Skill Standards for Biotechnology. The Austin Community College Biotechnology Program has formally adopted and applied these standards to its program and is recognized by the Texas Skill Standards Board (). Each course in the Biotechnology Program fulfills a specific set of skill standards. Those can be found here:

The skill standards applied to Introduction to Biotechnology (BIOL1414) are shown below.

Texas State Skills Standards (TSSB) Key Activities for BIOL 1414 Introduction to Biotechnology

A3 Operate equipment

B2 Perform assays and experiments

C4 Suggest continuous improvements

C5 Coordinate with work team

B3 Troubleshoot experiments and equipment C7 Handle and dispose of hazardous materials

B4 Perform B5 Communicate data analysis results

C8 Maintain D1 Maintain lab

security

notebook

C1 Participate in employersponsored safety training

D Create documents

C3 Identify unsafe conditions and take corrective action

Introduction to Biotechnology ? Course Competencies The ACC Biotechnology Program has been designed to match the needs of the biotechnology job market in our immediate area. We have invited industrial partners from our community to contribute to the competency goals for each course, including this introductory course, to assure that our students are adequately prepared for positions in their companies. The following list describes the areas of expertise that you will be introduced to in this course, and may provide you with an organizational plan for the archiving of your records in your notebooks for this course. As you progress through the ACC Biotechnology Program, you can add to these archives as you build on the basics learned in this introductory course.

Basic operations in the laboratory Purpose: There are special approaches and precautions that must be taken in any biological laboratory. This includes procedures for safe handling and storage of hazardous chemicals and biologicals. Also, the special methods for setting up and following detailed protocols are emphasized, as well as methods for recording and archiving results properly. Includes: Safety in the Laboratory Math Skills for the Laboratory Documentation and the Lab Notebook Molar Solutions and Dilutions Appendices

BIOL1414 Lab Manual Fall 2011

8

 ................
................

In order to avoid copyright disputes, this page is only a partial summary.

Google Online Preview   Download