BIO 510 RECOMBINANT DNA TECHNIQUES LABORATORY
Dept of Biology
College of Arts and sciences
Lab: MW 2:00 – 4:50 p.m., 224 T.H. Morgan
Lecture: F 2:00 – 2:50 p.m., 109 T.H. Morgan
Instructors:
Dr. Brian Rymond, 335A T.H. Morgan Building, 257-5530, rymond@uky.edu (preferred)
Office hours Tuesday and Thursday 9:15 - 10:15 AM (or by appointment).
Dr. Pete Mirabito, 302 T.H. Morgan Building, 257-7642, pmmira00@uky.edu (Preferred) . Office hours
Tuesday and Thursday 9:00 - 10:00 AM (or by appointment).
Assistants: Min Chen, 335 THM (chen.min@uky.edu); Dustin Perry 301A THM (dwperr2@uky.edu)
Bulletin Description
An introduction to the construction, isolation, and analysis of recombinant DNA clones, with emphasis on
practical experience in basic techniques. Graduate students will be given first preference in course
enrollment. Lecture, one hour; laboratory, 6 hours per week. Prereq: BIO 304 and BIO 315 or equivalent
with consent of instructor.
Course Content/ Overview: This four-credit course will familiarize the advanced undergraduate and the
beginning graduate student with the theory and application of recombinant DNA technology. Emphasis is
placed on learning though direct experimentation. The techniques and skills acquired (e.g.,
prokaryotic/eukaryotic cell transformation, DNA isolation and subcloning, Cre-Lox mediated DNA
transfer, in vitro mutagenesis, DNA sequencing, RNA analysis, PCR, phenotypic selection, in vitro
transcription - among others) are broadly applicable in modern medical, industrial and basic biological
research.
Prerequisites: This advanced course has requirements for previous coursework in GENETICS [BIO 304
or equivalent] and CELL BIOLOGY (BIO 315 or equivalent]. Students are expected to apply genetic and
biochemical principles in their understanding of recombinant DNA technology. Time does not permit the
review of basic genetic or biochemistry principles for BIO 510.
Required Textbook & Background resources: Principles of Gene Manipulation and Genomics,7 th
Edition S.B. Primrose, R.M. Twyman (Blackwell Science Press, 2006). Laboratory exercises will be
provided by the instructor in class as a protocol set. The Cold Spring Harbor Press web site
(http://www.cshlpress.com/) is a particularly rich source of excellent reference manuals on PCR techniques,
molecular genetics, bioinformatics, and other topics related to molecular biology. Good books for
background reading include GENES VII (B. Lewin, Oxford Press, 2000), Molecular Biology of the Cell
(B. Alberts et al., Garland Press, 2002); Genomes (T.A. Brown, Garland Science, 2002), Biochemistry
1
(Berg et al., W. H. Freeman & Co., 2002 and Modern Genetic Analysis (Griffiths et al., 1999). These (and
related books) are available free online at:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=books .
Many other relevant books can be found at the UK library. Additional Materials: Each student will
receive a copy of New England BioLabs (NEB) product and resource catalog. The NEB catalog contains a
wealth of information on the application of the enzymes and reagents used in a molecular biology
laboratory. In addition, the book contains multiple appendices with practical information (genotypes of
bacterial strains, restriction maps, etc.) and techniques. It is well referenced throughout with primary
literature citations. Each student will also have access to a hard copy and PDF-file copy of the Users
Manual for Invitrogen’s Vector NTI version 10.3. Lap top computers with Vector NTI 10.3 will be
provided to you for use in class. Lap tops can be made available to you at times outside of class time, but
you must contact the TAs to arrange to sign out and sign in a lap top computer. Furthermore, you must use
the lap tops in the class room. You will also receive a single-use student license for Vector NTI for you to
install on your own personal computer (instructions will be provided in class). This version of the software
is for PCs only. If your computers are exclusively MACs, then you will need to arrange to use the BIO 510
lap tops to complete homework assignments and the take-home Exam 2. PLEASE NOTE that quizzes
and exams will include information presented in lectures, lab handouts, homework assignments, and
assigned readings (textbook, NEB catalog, others assigned in class). A lab coat must be worn at all
times in the laboratory. Please have it on before the start of the lab.
Web site links:
First Half Semester
http://web.as.uky.edu/Biology/faculty/rymond/BIO%20510/BIO%20510%20%20%20Recombinant
%20DNA%20Techniques%20Lab.htm
Second Half Semester www.tinyurl.com/bio5102009
Grading Policy: Your grade will be assigned based on your performance in:
Exam 1
25%
Exam 2
25
Quizzes or homework
30
Notebook
10
Class participation
10
--------------------100%
The first half of BIO 510 emphasizes techniques and approaches whereas the second half of the course
involves application of techniques and experimental design using Vector NTI computer software.
Performance in each half of the course will be evaluated appropriately. Questions on quizzes and
examinations may include essays, short-answer questions, objective questions, or output from Vector NTI.
One or more quizzes may be “open book” and require you to use your New England Biolabs catalog or the
Principles of Gene Manipulation and Genomics text in class. Homework exercises will be announced in
2
class or listed in the lab manual or on the course web site; unless otherwise noted all homework is due one
week from the assignment date. All students are expected to accurately record and analyze the laboratory
exercises in a laboratory notebook. The lab manual may be used instead of a separate lab notebook for the
first half of BIO 510; please use blank sheets (or the back of protocol pages) to record you data. Use a
loose-leaf notebook for the second half of the semester. The notebook will be graded twice, once on
October 15th and again at the end of the semester but it needs to be available for inspection throughout the
semester.
Graduate students in this course will be required to complete two additional homework assignments each
semester; one in the first half of the semester and one in the second half. The undergraduates have the
option of completing these assignments as well and dropping two other homework assignment (but not quiz
grades) from the average quiz + homework grade calculation. Note that only one homework assignment
can be dropped per half semester (that is, one when Dr. Rymond is instructor and one when Dr. Mirabito is
instructor).
Numerical Grade
Letter Grade
100-90
A
89-80
B
79-70
C
69-60
D (not available for graduate students.
For Graduate students 69% or below = E)
<60
E
Midterm grades (based on criteria in syllabus) will be posted by October 15 (after Exam 1)
Attendance Policy: Attendance is mandatory. At a minimum, multiple (2 or more) unexcused absences
(as defined by the UK University Bulletin) will lower your final grade by one letter.
Plagiarism and Cheating Policy: The University of Kentucky recently revised its rules on plagiarism and
other forms of academic cheating. Infractions of these rules may result in serious consequences, including
but not limited to receiving a failing grade of “E” for this course. A full description of this policy,
implementation procedures, and outcomes can be obtained at: http://www.uky.edu/Ombud/Plagiarism.pdf
The textbook – what it does and does not do. This textbook provides a general overview of virtually all
the techniques and approaches we use in the laboratory. It also includes many scientific citations of
historical interest and references relevant to technical innovations and other topical, directed reading. The
text helps bring our exceptionally diverse enrollment up to a common level of understanding and is a useful
3
starting point for our lectures and discussions. The textbook is not a lab manual and is not meant to explain
the lab exercises. In addition, this textbook is not meant to replace the primary literature in your scientific
education.
Lab order and intent (first half). Many protocols in molecular biology are of the “hurry up and wait” type
where one step in a protocol (e.g., cell transformation) is initiated then requires hours (or days) to “finish”.
Since a major goal of the lab is to have students lean and apply as many molecular biological approaches as
possible within the time constraints of the lab we usually work on several different lab exercises in
parallel. That is, we carry our “parts” of several experiments in each lab meeting. The flow chart shown
below provides an overview of what we hope to accomplish. A central “theme” of this half of the semester
might be termed “characterization of a eukaryotic gene”. The most important goal, however, is for you to
learn recombinant DNA technology. With this in mind, we also include number of experiments that teach
important concepts/techniques but do not directly relate to the characterization of the DNA provided in our
first working lab.
Student participation. The success of this class depends upon your active participation. Your opinions
are valued, please be an active participant - ask questions, present your data to your lab partners/the
TAs/instructors, discuss your own lab experiences & professional goals, suggest lab protocol modifications,
consider alternative approaches. We use three approaches to enhance student interaction during the first ½
semester. These are the Q&A strips (see below), a one-on-one instructor conference, and a homework
assignment to “design a lab” for next year’s class. Other ideas to encourage participation are welcome.
Question and answer (Q & A) strips are to be submitted the day of participation (explained in class);
please print your name clearly. No Q&A strips will be accepted after October 13 th. Question and answers
must be directed to Dr. Rymond verbally in class and must be 1) clearly formulated to raise or answer a
substantive point of discussion, and 2) spoken loudly enough to be heard by the entire class. No simple
(yes, no, why, I don’t know) type responses/questions will be accepted. Each question and answer strip will
count equally and will be summed to form the class participation grade in the first half of the semester.
Undergraduates cannot use this as one of their “dropped” homework assignments.
Meet with the instructor: I will schedule one office visit for each student during the semester. The
purpose of this visit is to gauge student learning and to provide feedback opportunities. Attendance at this
meeting will be scored as one Q & A strip.
Policy on academic accommodations due to disability. If you have a documented disability that requires
academic accommodations, please see me as soon as possible during scheduled office hours. In order to
receive accommodations in this course, you must provide me with a Letter of Accommodation from the
Disability Resource Center (Room 2, Alumni Gym, 257‐2754, email address jkarnes@email.uky.edu) for
coordination of campus disability services available to students with disabilities.
4
Tentative Class Schedule
BIO 510, Topic I
Molecular Characterization of a Eukaryotic Transcription Unit.
Reading assignments. The reading should be done before the start of class on the assigned day. You do
not have to memorize the tables & graphs assigned in the NEB catalog but you will need to know how to
use these as tools for homework or in open book exam segments. Homework is due one week after the
assignment is made. GRADED HOMEWORK ASSIGMENTS are in bold. Note that the quizzes and
exam will cover the lab assignments & discussions, required readings & homework, and the Friday
lectures. Reading assignments in addition to those listed below are found in the lab manual and others may
be assigned during the semester.
Tentative Class Schedule
Aug.
25: Course overview; lab group assignments, online safety training; basic lab technique.
1 Chemical Hygiene Plan/Laboratory Safetyhttp://ehs.uky.edu/classes/chemhyg/train.html
2 Hazardous Wastehttp://ehs.uky.edu/ehs_webquiz/quizzes/takequiz.asp?id=XKQOO%20%20
3 Fire Extinguisher Training-http://ehs.uky.edu/classes/fire/firetrain.html
4 Biosafety
http://ehs.uky.edu/classes/biosafety/class.html
Friday, August 27: Enzymes in molecular biology
30: LAB 1. Agarose gel fractionation & purification of DNA; double restriction endonuclease
digestion of vector DNA. Reading assignments: PDF files on Qiagen agarose gel recovery; New England
Biolabs (NEB) catalog pages 16, 52 (HindIII), 48 (EcoRI); 130-131; 170; 294-297 homework assignment
Sept:
1: LAB 2. Influence of DNA conformation on gel mobility; preparation of transformation
competent E. coli. EtBr vs SYBR safe; PDF files on pTZ19u map (print out for your notebook); 1 kbp
DNA ladder (print out for your notebook) map, SYBR safe; NEB 126-127; 183; 192; 334; 298-333, (know
how to read charts); Principles of Gene Manipulation & Genomics (PGMG) chapters 1-3 and Box 15.1 on
pages 310-311 homework assignment
Friday September 3: Restriction endonucleases: types, recognition sequences, reaction conditions, star
conditions, DNA methylation.
6: Labor Day – academic holiday
8: LAB 3. Joining DNA ends with T4 DNA ligase; isolation of total RNA from yeast; impact of
5
increased salt concentration on DNA migration; NEB 130-131; 152 (CIP); 203, 359 (Impact system); 335343; 346; 358 homework assignment
Friday September 10: Plasmid and viral cloning vectors PGMG chapter 4-5
13: LAB 4. E. coli transformation; determination of RNA yield and purity. Purification of a
recombinant protein, part I. NEB 151; 382-384; 344-345; 348-349; short quiz and homework assignment
15: LAB 5. Phenotypic characterization of recombinant DNA clones; fractionation of RNA by
denaturing gel electrophoresis; northern transfer. Purification of a recombinant protein, part II Riboprobe
manual PDF; homework assignment
Friday September 17:
Radiation Safety 2 lecture; (Radiation Safety Officer, Fred Rawlings)
20: LAB 6. Isolation of recombinant plasmid DNA from E. coli. Purification of a recombinant
protein, part III; Qiagen Miniprep PDF; Epicentre Yeast DNA isolation PDF; NEB 90-98
22: LAB 7. Analysis of recombinant clones; preparation of in vitro transcription template;
northern blot pre-hybridization; isolation of genomic DNA from Saccharomyces cerevisiae. Seraphin group
TAP web page (see course web site);
Friday September 24: Expression systems and cloning strategies PCR approaches for gene analysis and
modification; DNA sequencing. PGMG chapters 6, 8.
27: LAB 8. In vitro transcription; chromatographic separation of RNA and unincorporated;
ribonucleotide triphosphates; hybridization of northern blot with strand-specific probes. NEB 178-181; 163
(M13K07) homework assignment
29: LAB 9. Wash blots; autoradiography; preparation of single-stranded DNA template for DNA
sequencing. RNAi knockdown, Caenorhabditis elegans (part 1) PDF files on Realtime PCR and Icycler;
Friday Oct 1: RNAi technology; cloning and gene expression studies in yeast Realtime PCR PGMG
chapter 11 and pages 315-318.
Oct
4: LAB 10. Analysis of northern blot; quality check on single stranded DNA sequencing template;
RNAi-knockdown in C. elegans (part 2); PCR amplification of genomic DNA; inverse PCR plasmid
mutagenesis; assembly of template/primer hybrid; PDF file on Typhoon; PGMG chapter 7; short quiz
6: LAB 11. Analysis of PCR products; Real-time PCR; gene identification by in vivo
complementation. RNAi-knockdown in C. elegans (part 3). Affy hybridization and analysis PDF;
homework assignment
Friday October 8: DNA microarray technologies; bioinformatics. PGMG chapter 9
6
11: LAB 12. Analysis of DNA sequence files; completion of C. elegans experiment; finish up &
clean up, and review.
13: Exam 1 – during class period
Class Schedule Second half of BIO 510
Overview:
1. Knock-out an essential gene and a non-essential gene
2. Create conditional mutations in an essential gene by site-directed
mutagenesis
3. Create mutations in an essential gene by using error prone PCR (random
mutagenesis)
4. Delete a non-essential gene by using the Cre-lox site-specific
recombination system
5. Isolate whole chromosomes and analyze them using pulsed-field gradient
gel electrophoresis in combination with rare-cutting restriction enzymes an
with the RecA-Achilles Cleavage strategy
Friday, 10/17
Introduction to the 2nd Half of the course
Introduction to Vector NTI
Monday, 10/20
Start Gene Knock-out lab in vitro and in silico
Wednesday, 10/22
Continue Gene Knock-out lab in vitro and in silico
Friday, 10/24
Questions and answers regarding homework #1
Monday, 10/27
Continue Gene Knock-out lab; Start Site-directed mutagenesis lab in silico
Wednesday, 10/29
Start Site-directed mutagenesis lab in vitro
Continue Start Site-directed mutagenesis lab in silico
Friday, 10/31
Homework #1 due; Continue Start Site-directed mutagenesis lab in silico
Monday, 11/3
Finish non-essential gene Knock-out lab;
Continue essential gene Knock-out lab;
Start Site-directed mutagenesis lab in vitro
Wednesday, 11/5
Finish essential gene Knock-out lab;
Continue Site-directed mutagenesis lab in vitro
Friday, 11/7
Questions and answers regarding homework #2;
Continue Site-directed mutagenesis lab in silico
Monday, 11/10
Continue Site-directed mutagenesis lab;
Start the Random Mutagenesis lab
7
Wednesday, 11/12
Finish Site-directed mutagenesis lab;
Continue the Random Mutagenesis lab
Friday, 11/14
Questions and answers on homework #2:
Continue Site-directed mutagenesis lab in silico
Monday, 11/17
Continue Random Mutagenesis lab;
Start the Cre-lox deletion lab work
Start the Whole Chromosome Analysis lab work
Wednesday, 11/19
Continue Whole Chromosome Analysis lab;
Start Cre-lox deletion lab using Vector NTI
Friday, 11/21
Homework #2 due;
Continue labs in Vector NTI
Monday, 11/24
Continue Random Mutagenesis lab
Continue the Whole Chromosome Analysis lab work;
Finish Cre-lox deletion lab
Wednesday, 11/26
Friday, 11/28
NO MEETING – Thanksgiving vacation
NO MEETNIG – Thanksgiving vacation
Monday, 12/1
Finish Random Mutagenesis lab;
Continue Whole Chromosome Analysis lab work
Wednesday, 12/3
Continue Whole Chromosome Analysis lab work
Friday, 12/5
Final session on Vector NTI
Monday, 12/10
Finish Whole Chromosome Analysis lab work
Wednesday, 12/12
Day to catch-up & clean up and TURN IN NOTEBOOKS
Friday, 12/14
Take-home Exam Distributed and explained
Friday, 12/19
EXAM 2 ANSWERS DUE TO ME BY EMAIL by 8 AM
Course Goals
The student will develop an appreciation and understanding of the theory and applications of recombinant
DNA technology.
The student will become familiar with standard bioinformatic tools used to design and analyze DNA
molecules.
The student will develop an understanding of the methods and processes of scientific inquiry, with
emphasis on molecular analyses of DNA, RNA, and protein.
The student will appreciate the importance of molecular genetic analyses in modern medical, industrial, and
basic biological research.
The student will be prepared for graduate studies in molecular genetics.
The students’ needs will be met by fostering the development of critical thinking, reasoning, and problem
solving skills.
8
Student Learning Outcomes Objectives
The Scientific Method applied to Recombinant DNA Technology and Molecular Genetics
1. Demonstrate an understanding of the role of the scientific method in molecular genetic analyses.
2. Interpret raw molecular genetic data to synthesize testable hypotheses for the structure and function of
DNA molecules.
3. Demonstrate an understanding of molecular maps of DNA and be able to use these maps to predict the
outcome of biochemical manipulations of DNA.
Organizing Concepts
1. Demonstrate the procedures used to clone and characterize a DNA molecule.
2. Demonstrate the procedures use to determine the in vivo function of a DNA molecule.
3. Demonstrate the procedures used to determine the RNA and protein coding capacity of a DNA molecule.
Creating and Analyzing Recombinant DNA Molecules
1. Describe the structure and function of bacterial cloning vectors.
2. Contrast the roles of genetic selection and genetic screens in cloning DNA molecules.
3. Describe the function of enzymes used in cloning DNA molecules and in the analysis of those clones.
4. Demonstrate how gel electrophoresis is used in the creation and analysis of Recombinant DNA
molecules, the size fractionation of RNA, and the characterization of proteins.
6. Demonstrate the procedures for the preparation of nucleic acid templates for DNA sequencing and the
analysis of DNA sequence output files
Analyzing the Coding Capacity of Cloned DNA Molecules
1. Demonstrate northern blot analysis and discuss the importance of base pairing to northern blotting.
2. Demonstrate real time PCR and compare and contrast real time PCR with Southern and northern blotting
for the identification of DNA polymorphisms.
3. Demonstrate how to use informatic tools to predict the coding capacity of a DNA molecule, the
secondary structure of RNA and the structural motifs of proteins.
4. Demonstrate western blot analysis and discuss the role of antibodies in western blotting.
5. Demonstrate how gene and protein fusions and affinity purification can be used to purify a recombinant
protein.
6. Demonstrate how gene complementation can be used to score for biological function.
7. Demonstrate how RNAi is used to score for biological activity
Using in vitro Mutagenesis Techniques to Dissect the in vivo Function of a DNA Molecule.
1. State the principles behind the design of gene knock-out constructs.
2. Demonstrate the role of homologous recombination in creating gene knock-outs.
3. Describe the approaches used to conduct gene knock-outs in different model organisms.
4. Demonstrate how to use informatic tools to identify target sequences for site-specific mutagenesis.
5. State the principles behind the design of gene knock-in constructs.
Homework Assignments and Plagiarism
CUT and PASTING or other direct quotation from web sites or other sources – including lab partner
or others.
When is it allowed and appropriate? When reporting the output of a Web-based computer program you
ran such as BLAST, or the RNA folding programs, or when providing a data in the form of a figure or table
from the primary literature (that is, a journal article). In last case, the full literature citation must also be
provided.
Otherwise, when is it allowed? Almost never – the one exception that comes to mind is copying the
literature citation itself, e.g., Wang, Q., He, J., Lynn, B. and Rymond, B.C. 2005. Interactions of the yeast
SF3b splicing factor. Mol. Cell Biol, 25: 10745-10754. Unless otherwise directed, use scientific journal
9
citations only (not textbook, Wikipedia, or sales materials).
In all other cases, your response to a question should be your own intellectual output. Using search
engines to find relevant information is fine, but simply transferring such output from machine to paper is
not the goal. You are being asked to read, integrate, and respond in your own words – showing that you
understand the issue under question, not simply that you are adept in keystrokes and database mining.
Direct, unattributed quotations represent plagiarism, are unethical and may be illegal. The first
instance of plagiarism will result in a grade of “0” on the assignment. Additional instances of
plagiarism will be treated much more seriously and could even result in dismissal from the
University, see: http://www.uky.edu/StudentAffairs/Code/part1.html
and also:
http://www.uky.edu/Ombud/Plagiarism.pdf
Direct, attributed quotations (i.e., with citations) are appropriate only if I ask for direct, attributed
quotations – otherwise, everything should be in your own words.
Course Policy on Classroom civility and decorum: The university, college and department has a
commitment to respect the dignity of all and to value differences among members of our academic
community. There exists the role of discussion and debate in academic discovery and the right of all to
respectfully disagree from time-to-time. Students clearly have the right to take reasoned exception and
to voice opinions contrary to those offered by the instructor and/or other students (S.R. 6.1.2). Equally,
a faculty member has the right -- and the responsibility -- to ensure that all academic discourse occurs
in a context characterized by respect and civility. Obviously, the accepted level of civility would not
include attacks of a personal nature or statements denigrating another on the basis of race, sex, religion,
sexual orientation, age, national/regional origin or other such irrelevant factors.
Course policies on excused absences/ make-up opportunities / documentation:
Make-up of work/exams will only be permitted for excused absences as defined by University Senate Rules
WRITTEN SUPPORTING DOCUMENTATION regarding a missed exam / class activity MUST be
presented to the course instructor within a week after a student returns to class after the excused absence
otherwise an automatic score of zero will be earned for the activity/exam. Contact the Instructor regarding
make-up activities/exams.
10