BITC1311 Introduction to Biotechnology - Austin Community College

BITC 1311 Introduction to Biotechnology
Laboratory Manual
Seventh Edition
Fall 2008
Linnea Fletcher, Evelyn Goss, Patricia Phelps, and Angela Wheeler
ISBN: BITC1311008
Table of Contents
Introduction 3 Safety in the ACC Laboratory 7 Math Skills for the Laboratory
13 Documentation and the Lab Notebook
21 Basic Tools in the Biotechnology Laboratory
24 Using a Micropipetter 29 Calibrating Lab Instruments 33 Molar Solutions and Dilutions
38 RNA Isolation 45 Transformation of E. coli
52 Plasmid Isolation 56 Restriction Enzyme Mapping of DNA 59 Green Fluorescent Protein Purification
67 Protein Electrophoresis of GFP Samples
71 DNA Fingerprinting by Alu PCR
77 Bioremediation: Environmental Clean-Up
83 DNA Fingerprinting by Southern Blot
87 ELISA for HIV 92 Bioinformatics 97 Appendix A: ACC Lab Safety Procedures
116
Appendix B: Hints for Solving Numerical Problems
122
Appendix C: Summary of Chemical Hazards, MSDS, Chemical Labels and
Solution Prep Forms 124
Appendix D: Graphing Data 130
Appendix E: Summary of Good Laboratory Practices
133
Appendix F: Agarose Gel Electrophoresis with Ethidium Bromide
136
Introduction
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. To help you to develop an archiving system for your records, it is
recommended that you purchase two 3-ring binders or one 3-ring binder and a
bound notebook for this course. Other materials required for this course include 1. Personal protective equipment (PPE): goggles and a lab coat
(recommended)
2. Personal equipment: fine-point Sharpie markers Before you can begin working in an ACC teaching 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.
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. Some statistics about biotechnology reflect the
expansion of this industry: (found at www.bio-link.org in the year 2004)
More than 325 million people worldwide have been helped by the more than
130 biotechnology drugs and vaccines approved by the US Food and Drug
Administration (FDA). Of the biotech medicines on the market, 70 percent
were approved in the last six years.
There are more than 350 biotech drug products and vaccines currently in
clinical trials targeting more than 200 diseases, including various
cancers, Alzheimer's disease, heart disease, diabetes, multiple sclerosis,
AIDS and arthritis.
Biotechnology is responsible for hundreds of medical diagnostic tests that
keep the blood supply safe from the AIDS virus and detect other conditions
early enough to be successfully treated. Home pregnancy tests are also
biotechnology diagnostic products.
Consumers already are enjoying biotechnology foods such as papaya, soybeans
and corn. Hundreds of biopesticides and other agricultural products also
are being used to improve our food supply and to reduce our dependence on
conventional chemical pesticides.
Environmental biotechnology products make it possible to clean up hazardous
waste more efficiently by harnessing pollution-eating microbes without the
use of caustic chemicals.
Industrial biotechnology applications have led to cleaner processes that
produce less waste and use less energy and water in such industrial sectors
as chemicals, pulp and paper, textiles, food, energy, and metals and
minerals. For example, most laundry detergents produced in the United
States contain biotechnology-based enzymes.
DNA fingerprinting, a biotech process, has dramatically improved criminal
investigation and forensic medicine, as well as afforded significant
advances in anthropology and wildlife management.
There are 1,457 biotechnology companies in the United States, of which 342
are publicly held.
Market capitalization, the total value of publicly traded biotech companies
at market prices, was $224 billion as of early May 2002.
The biotechnology industry has more than tripled in size since 1992, with
revenues increasing from $8 billion in 1992 to $27.6 billion in 2001.
The U.S. biotechnology industry currently employs 179,000 people; that's
more than all the people employed by the toy and sporting goods industries. Biotechnology is one of the most research-intensive industries in the
world. The U.S. biotech industry spent $15.6 billion on research and
development in 2001.
The top five biotech companies spent an average of $89,400 per employee on
R&D in 2000. The biotechnology industry has also been steadily growing in the Austin
area. Today, Austin's bioscience community encompasses approximately 85
companies that produce products and services such as pharmaceuticals,
preventive medicines, medical devices, laboratory tools and analysis, and
gene based cancer therapies. 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 Center in nearby
Bastrop.
Biotechnology Techniques and Skills Included in This Course 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. 1. 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
Appendix A: ACC Lab Safety Procedures
Appendix B: Hints for Solving Numerical Problems
Appendix C: Summary of Chemical Hazards, MSDS etc
Appendix D: ACC Hazardous Waste Program etc
Appendix E: Graphing Data
Appendix F: Summary of Good Laboratory Practices
Appendix G: Agarose Gel Electrophoresis with Ethidium Bromide 2. Instruments and Equipment
Purpose:
An important part of working in any laboratory is the proper use and
calibration of instruments and equipment. You will become familiar with
general information about the use of lab equipment, as well as more
detailed information about the step-by-step procedures for the specific
instruments that you use.
Includes:
Basic Tools in the Biotechnology Laboratory ACC Biotech
Program Equipment locator
Using a Micropipetter micropipetters
Calibrating Lab Instruments balances and pH meters
Restriction Enzyme Mapping of DNA agarose gel
electrophoresis
GFP Chromatography denaturing polyacrylamide
gel electrophoresis
DNA Fingerprinting: Alu PCR thermal cycler,
agarose gel electrophoresis 3. Working with DNA and proteins
Purpose:
It is important to be familiar with the basic techniques for purifying
and analyzing biomolecules. You will learn to isolate, digest, and
analyze DNA, as well as transform E. coli with a recombinant plasmid.
You will also learn some basic methods to purify and analyze proteins.
Includes:
Transformation of E. coli in vivo
amplification of plasmid