Instrumentation and Measurement in Biotechnology
ISAT 305, Fall 2001
James Madison University
College of Integrated Science and Technology
Dr. Robert McKown Dr. Rita Teutonico
Office: ISAT 326 Office: ISAT 352
Phone: 568-2776 Phone: 568-2735
E-mail: firstname.lastname@example.org E-mail: email@example.com
Dr. Ron Raab Dr. George Coffman
Office: ISAT/CS 327 Office: ISAT 318
Phone: 568-2729 Phone: 568-2767
E-mail: firstname.lastname@example.org E-mail: email@example.com
Catalog Course Description
ISAT 305. Instrumentation and Measurement in Biotechnology (0,2). 1 credit.
This course provides a hands-on experience of the techniques and instrumentation used in
the modern biotechnology laboratory. Topics include aseptic techniques for establishing
microbial cultures, detection and analysis of recombinant DNA molecules, protein
purification, SDS gel electrophoresis, and the use of PCR technology for genetic analysis.
Prerequisite: ISAT 300 and ISAT 350, or permission of instructor.
Statement of Purpose
Welcome to ISAT 305, Instrumentation and Measurement in Biotechnology. The
purpose of this course is to provide you a hands-on learning experience in a laboratory
setting that focuses on techniques used in biotechnology. You will learn how to maintain
a laboratory notebook, perform routine laboratory duties, execute experimental protocols,
and operate laboratory equipment used in biotechnology procedures. The areas of focus
include sterile techniques, cell culturing, DNA purification, DNA restriction analysis,
cloning, PCR genetic analysis, DNA sequence analysis, protein purification, and SDS
polyacrylamide gel electrophoresis. As a class, we will discuss how these laboratory
techniques are used in the biotechnology industry.
Requirements and Learning Resources
An ISAT 305 Course Pack is required and available for purchase at the CISAT Copy
Center. A Laboratory Notebook and safety glasses are required (these can be purchased
from the JMU bookstore or the CISAT Copy Center). The course grade will be
determined from grading the lab notebooks, computer exercises, and problem sets.
First notebook grading 100
Second notebook grading 100
Problem sets and assignments 150
After the successful completion of this course, students should be able to:
Make calculations and prepare stock laboratory solutions.
Perform sterile techniques and operate an autoclave.
Purify and maintain bacterial strains on agar plates.
Culture pure microbial strains in liquid media.
Clone DNA sequences into bacterial plasmids
Transform bacteria with recombinant DNA plasmids
Purify recombinant plasmid DNA from bacteria.
Perform agarose gel electrophoresis to separate DNA fragments.
Analyze and map recombinant DNA fragments.
Set up PCR reactions and operate a thermal cycler.
Analyze PCR reaction products and calculate genetic frequencies.
Analyze DNA sequence information.
Purify proteins using column chromatography
Determine protein concentrations
Analyze proteins by SDS polyacrylamide gel electrophoresis..
Maintain a laboratory notebook according to biotechnology industry standards.
Laboratory Notebook Guidelines
This course is divided into two major sections (Recombinant DNA Analysis and Human
Genetic Analysis/Protein Purification). Each section contains multiple interrelated
laboratory exercises that constitute a continuous line of experimental inquiry. Laboratory
notebooks will be turned in for grading after completion of each section.
Leave 2 pages for a table of contents.
Number each page.
Use permanent ink for all entries.
Securely attach and label all data entries (graphs, tables, and charts).
Sign, witness, and date each page (use the stamp provided).
Each exercise will have a brief description of the purpose (what are you doing?).
Each exercise will have a description of materials/methods (how was it done?).
Certain protocols can be taped into the notebook or referenced.
Each exercise will have all the data generated during the lab (what happened?).
Each section will have a description of the purpose for the entire section (what is the
purpose of all the exercises in this section?).
Each section will have a summary of all the relevant data and results generated
including any calculations, graphs or tables.
Each section will have a discussion and conclusions of the final results.
All exercises and sections will provide documentation for any references used.
Notebooks should be neat, well organized, and readable.
ATTENDANCE IS REQUIRED
NO MAKEUP LABS
The classroom attendance policy, laboratory safety, and collaboration guidelines for each
section will be discussed at the first lab meeting.
Outline of Laboratory Exercises and Assignments
Week Exercise Laboratory Exercises and Assignments .
8/27 1 Course introduction, laboratory safety, and lab notebooks
9/3 2 Cloning of lambda DNA into pUC19:
Restriction endonuclease and ligation reactions
Biotechnologist Exercise 1: Image Analysis
Problem Set 1 Due (20 points)
9/10 3 Transformation of E. coli with recombinant DNA
Biotechnologist Exercise 2: Bacterial Transformation
Biotechnologist Exercise 1 Due (10 points)
Problem Set 2 Due (10 points)
9/17 4 Purification of plasmid DNA
Biotechnologist Exercise 2 Due (10 points)
9/24 5 Restriction digestion of plasmid DNAs
Biotechnologist Exercise 3: Restriction Mapping
Problem Set 3 Due (10 points)
10/1 6 Agarose gel electrophoresis
Biotechnologist Exercise 4: Gel Electrophoresis
Biotechnologist Exercise 3 Due (10 points)
Problem Set 4 Due (10 points)
10/8 7 Introduction to PCR genetic analysis
Biotechnologist Exercise 5: Polymerase Chain Reaction
Biotechnologist Exercise 4 Due (10 points)
Turn in lab notebooks for grading (100 points)
10/15 8 Isolation of human DNA
PCR amplification of human DNA
Biotechnologist Exercise 5 Due (10 points)
Problem Set 5 Due (10 points)
10/22 9 Agarose gel electrophoresis of amplified DNA
Biotechnologist Exercise 6. PCR in Medicine
10/29 10 Hardy-Weinberg genetic analysis of all PCR data
Biotechnologist Exercise 6 Due (10 points)
11/5 11 Purification of Green Fluorescent Protein
Biotechnologist Exercise 7. DNA Sequencing
Problem Set 6 Due (20 points)
11/12 12 GFP Quantitation and SDS PAGE for molecular weight determination
Biotechnologist Exercise 7 Due (10 points)
11/26 13 Completion of GFP MW determination and protein purification table
12/3 14 Course summary
Turn in lab notebooks for final grading (100 points)
Instrumentation and Measurement in Biotechnology
ISAT 305, Fall 2001
Cloning and Recombinant DNA Analysis
Purpose and Objectives
The purpose of this section is to provide you a hands-on learning experience in cloning,
transformation, and analysis of recombinant DNA. You will be cloning a fragment of
DNA from the bacterial virus lambda into the plasmid vector pUC19. The recombinant
plasmid DNA will be transformed into E. coli cells and grown on agar plates with a color
indicator. From a mixture of bacteria containing plasmid DNAs with a variety of
possible inserts (cloned fragments), you will select a single bacterial colony with a
recombinant plasmid DNA using a visual color selection strategy. A homogeneous
population of the bacteria containing your cloned candidate will be cultured to produce
enough plasmid DNA for analysis. The plasmid DNA will be purified from the bacteria,
cut with restriction enzymes, and analyzed by agarose gel electrophoresis. From this
analysis, you will generate a physical map of the recombinant plasmid DNA and
determine which fragment has been cloned.
Restriction digestion of lambda and pUC19 DNA
Ligation of lambda DNA into pUC19
Transformation of E. coli with recombinant plasmid DNA
Preparation of sterile media and agar plates
Selection of recombinant bacteria and purification to a single colony
Purification of recombinant plasmid DNA
Digestion of recombinant plasmid DNA with restriction enzymes
Agarose gel electrophoresis of digested plasmid DNA
Restriction mapping of recombinant plasmid DNA
Lambda DNA will be cut with the restriction enzyme EcoRI. The cloning vector pUC19
will be cut with the same restriction enzyme. The cut lambda DNA will be mixed with
the cut vector DNA and covalently bonded with DNA ligase. The ligated mixture will be
transformed into bacterial cells and spread onto an agar plate containing a color indicator.
The agar plate will contain a mixture of blue and white colonies. Blue bacterial colonies
do not contain a cloned lambda fragment and white colonies contain a plasmid with a
cloned lambda DNA fragment. From this agar plate, a single white colony (your
candidate) will be selected and streaked out on a fresh agar plate for further analysis. The
standard laboratory nomenclature of a small letter p followed by your initials and a
number will be used to name your candidates (for example, candidates for Bob Jones
would be named pBJ1, pBJ2, etc.). Your candidate will be grown in liquid culture media
and the plasmid DNA purified. You will select a number of restriction enzymes to digest
the candidate pDNA and separate these fragments by agarose gel electrophoresis. From
the resulting restriction patterns, you should be able to generate a restriction map of your
recombinant plasmid DNA and identify which fragment of lambda DNA was cloned.
DNA restriction maps of linear lambda DNA and circular pUC19 DNA will be provided.
Selection of Bacteria with Recombinant DNA using the pUC19 Plasmid Vector
The cloning vector pUC19 uses a blue/white color indicator to identify bacteria with
pDNA molecules containing a cloned insert. The important features of this vector (see
attached map) are:
1. An ampicillin resistance gene for selection.
2. A functional laci gene.
3. The lactose promoter/operator which is repressed by the product of the laci gene and
induced by lactose or IPTG.
4. A lacZ gene under control of the lactose promoter/operator that produces the enzyme
5. A multiple cloning site within the lacZ gene.
6. An origin of replication.
This vector was designed such that the multiple cloning site does not disrupt the enzyme
activity of B-galactosidase. When this vector is present in bacterial cells, the lacZ is not
expressed because the laci gene product (repressor) is bound to the lactose
promoter/operator region. In the presence of lactose or the chemical IPTG, the laci gene
product does not bind to the promoter/operator and B-galactosidase is made. X-gal is a
histochemical substrate for B-galactosidase. Bacteria containing an active B-
galactosidase cleave this substrate producing a blue color. Bacteria that do not contain an
active B-galactosidase can not cleave X-gal and it remains white.
In a cloning reaction that uses a single enzyme to cut the vector, most of the plasmids will
reform without a cloned insert when DNA ligase is added. Only a small percentage of
cut plasmids will receive an insert before ligation occurs. Without some type of selection
process, many individual pDNAs must be analyzed to find the one with an insert. The
pUC selection system was designed to visually identify bacteria containing a plasmid
DNA with a cloned insert.
The selection process works on the notion that an insert into the multiple cloning sight of
the coding sequence of the lacZ gene will yield an inactive B-galactosidase enzyme from
the introduction of a “stop codon” within the insert or the generation of an inactive fusion
protein. The ligated mixture of vector and insert is transformed into bacteria and the
bacteria is spread on an agar plate containing ampicillin, IPTG, and X-gal.
Transformants without an insert will produce B-galactosidase, cleave X-gal, and produce
blue colonies. Transformants with an insert will not produce an active B-galactosidase,
will not cleave X-gal, and produce white colonies. Individual white colonies can then be
visually selected from a field of blue colonies.
DNA restriction Mapping
Using the banding patterns visualized on agarose gels, a circular plasmid map can be
generated. This is usually a trial and error process in which several maps are drawn and
the one that agrees with the data is selected. In some cases, the orientation of the cloned
DNA relative to the plasmid can be determined.