Biology 13 SYLLABUS
Winter 2015
Biology 13
Gene Expression and Inheritance
Professor
Prof. Tom Jack
LSC 331
Office Hours: Tu 10-11:30, W 4-5, Th 1:30-3
Instructor
Natalia Vecerek
LSC 123
Office Hours: Th and Sunday, 7-9PM in 113 Silsby
Graduate Teaching Assistants
Mu A, Lita Bozler, Balint Kacsoh, Frederick Varn, Chris Giaque, Ofelia Tacchelly
Laboratory Instructors
Nick Sylvain (Laboratory Director)
Cori Anderson
Lara Park
Prerequisites
There are no enforced prerequisites for Biology 13. However, Biology 11 or a strong prior preparation
in biology is recommended. The details of Biology Department’s recommendations for entry into
Biology 13, for those that have not taken Biology 11, can be found at
https://canvas.dartmouth.edu/courses/5105/pages/how-to-interpret-the-score-on-the-placement-slashadvisory-test.
Textbook
iGenetics, A Molecular Approach by Peter Russell, 3rd edition.
Course Goals
At the end of the course, students will:
• understand the “central dogma” of molecular biology, i.e. the key gene products and molecular
mechanisms responsible for the transfer of genetic information from DNA to RNA to protein
and ultimately to the expression of a phenotype
• understand how genetic information is recombined and transmitted from one generation to the
next
• understand the fundamental concepts that underlie the regulation of the expression of genetic
information
• be familiar with specific foundational experiments and well-studied examples in molecular
genetics
• be able to think critically and solve problems in genetics and molecular genetics
• be capable of analyzing different types of data (from genetic crosses or genomic analysis) to
determine genetic linkage and to create a genetic map
• be able to investigate a current problem in genetics and effectively communicate key scientific
information to others
Office hours
Both Natalia and I will be having several hours of office hours per week. We have tried to schedule
office hours at different times of day and different days of the week to accommodate varying student
schedules.
Special appointments
If you have particular concerns, difficulties or interests that you would like to discuss individually,
email to set up an appointment.
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Biology 13 SYLLABUS
Winter 2015
Study groups
We encourage students to form their own study groups. The Academic Skills Center will be organizing
study groups for Bio 13, led by a student who has previous taken Biology 13. The sign up for study
groups will be during the second week of the course.
Canvas
Course materials for Biology 13 will be available in Canvas. The syllabus, announcements, reading
assignments, Powerpoint class presentations, pre-lecture screencasts, solutions to problem sets and
exams, information about the laboratory etc. will be posted in Canvas.
Interactive Technology
We will use an interactive technology, called “Lecture Tools”, that will allow you to respond to a
variety of different types of questions posed in class. To take best advantage of Lecture Tools, you
should bring your computer or iPad to class. When the class responds to one of the interactive
questions, we can immediately see the results for the entire class. Class participation using Lecture
Tools will count for a small percentage of your overall grade. With the Lecture Tools questions, the
key is to participate; your participation grade is not dependent on answering questions correctly. To get
full credit for Lecture Tools participation, you need to respond to questions in 85% of the class
periods, and complete 85% of the pre-class exercises. In other words, if you forget your computer, or
are sick and have to miss a class, or are too busy to complete the pre-class exercises, you will not be
penalized until you have missed more than 15% of classes (>5 classes) or failed to complete more than
15% of the pre-class exercises (>5 assignments).
Assessment of your academic performance
First Exam
Second Exam
Third Exam
Fourth Exam
Final Exam
Project
Participation (pre-lecture and in class)
Laboratory
10%
10%
15%
15%
20%
5%
5%
20%
We will have four exams during the term, roughly every two weeks, and in total, these exams will
count for 50% of your grade. The final exam will cover all topic areas (i.e. it is cumulative), and will
count for 20% of your final grade. In general, the final exam will be less detailed than the mid term
exams, and will emphasize major concepts, integration and synthesis. 5% of your grade will be based
on a project that we will undertake in the last week of the course. This project will involve reading
and presenting a paper from the primary literature. 5% of your grade will be for participation, both for
completing the pre-class material and being prepared for and participating in class discussions,
problem sessions, and in-class interactive questions. The remaining 20% will be based on
performance in the laboratory component of the course.
Academic Honesty
Academic honesty is essential. The following is quoted directly from the Dartmouth College Student
Handbook: "Students who submit work that is not their own or who commit other acts of academic
dishonesty forfeit the opportunity to continue at Dartmouth." The complete text of the Academic
Honor Principle is available at http://www.dartmouth.edu/~uja/honor/. Please read it carefully; you are
responsible for it. In Bio 13, where assessment is based on in-class exams and a final exam, the
application of the Honor Principle is quite simple; all your quiz and exam work must be 100% your
own, and you may not use any unauthorized notes, textbook, electronic resources (smart phones, iPads,
laptops, internet) or other resources during the quizzes and exam. Any violations of the Honor
Principle within the context of Biology 13 will be referred to the Undergraduate Judicial Affairs Office
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Biology 13 SYLLABUS
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and can result in a hearing before the Committee on Standards and can result in your suspension for
multiple terms or, in the most extreme cases, separation from the College.
There are a number of situations in which a student in Biology 13 might be tempted to violate the
Academic Honor Principle. These situations include (but are not limited to) the following:
a) Examinations must be completed without reference to unauthorized written materials or
electronically accessed materials other than those provided with the exam paper and must be
completed without communication with anyone else (the only permissible exception is that students
may request clarification of any exam question from the course faculty and staff who are present
expressly for that purpose). The answers that you provide must be entirely your own work.
b) We allow re-submission of exams for potential re-grading by the professor. Any alteration of the
answers between the time when the graded papers were returned to the student and the time when the
paper was submitted for re-grading constitutes a breach of the Academic Honor Principle. To deter this
practice, we routinely photocopy exams after grading them.
c) Some laboratory exercises are performed in small groups, and we encourage collaborative analysis
of the data. However, any work submitted for grading must represent the original words of the student
submitting that report. Do not share computer files of work (including text, graphs, tables, etc.) to be
submitted for grading! The student misrepresenting the work of another as his or her own is in
violation of the Academic Honor Principle and it is quite possible that the Committee on Standards
might find the student providing the original file also to be in violation. Honesty is the foundation of
the academic pursuit of knowledge. In recognition of this, the faculty of Biology 13 will not overlook
any violations of the Academic Honor Principle. Indeed, the Faculty Handbook of Dartmouth College
states explicitly that College Faculty members are obligated to report potential violations of the
Academic Honor Principle to the Dartmouth College Committee on Standards. Should the Committee
on Standards find the student to be in violation of the Academic Honor Principle, punishments usually
involve suspension for multiple terms or separation of the student from the College.
Disabilities
We encourage students with disabilities, who may need disability-related classroom accommodations,
to make an appointment to see one of the professors as soon as possible, preferably before the end of
the second week of the term. All discussions will remain confidential, although the Student
Accessibility Services office may be consulted to discuss appropriate implementation of any
accommodation requested.
Religious observances
Some students may wish to take part in religious observances that occur during this academic
term. If you have a religious observance that conflicts with your participation in the course,
please meet with the professor before the end of the second week of the term to discuss appropriate
accommodations.
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Class Schedule
(check Canvas for revisions during the term)
Jan 5
Jan 7
Jan 8
Jan 9
Jan 12
Jan 14
Jan 15
Jan 16
Jan 19
Jan 21
Jan 22
Jan 23
Jan 26
Jan 28
Jan 29
Jan 30
Feb 2
Feb 4
Feb 5
Feb 6
Feb 9
Feb 11
Feb 12
Feb 13
Feb 16
M
W
Th
F
M
W
Th
F
M
W
Th
F
M
W
Th
F
M
W
Th
F
M
W
Th
F
M
Feb 18
Feb 19
Feb 20
Feb 23
Feb 25
Feb 26
Feb 27
Mar 2
W
Th
F
M
W
Th
F
M
Mar 4
Mar 5
Mar 6
Mar 9
W
Th
F
M
Course overview
DNA as Genetic Material, DNA Structure
Course study tips (led by Natalia)
Genomes/Chromosomes/Chromatin
DNA Replication
Transcription I
Transcription II
Exam #1 (in class)
Martin Luther King Day – no class
Transcription III
Genetic Code
Protein Synthesis - Translation
Mutation
Effects of Mutation
Genes and Gene Products
Exam #2 (in class)
Meiosis
Patterns of Inheritance I – dihybrid cross
Patterns of Inheritance II – deviations
Winter Carnival – no class
Patterns of Inheritance II – sex linkage
Linkage and Mapping I
Linkage and Mapping II
Pedigrees
In class review
Exam #3 – 7-10 PM
Mapping with Molecular Markers
GWAS, and QTLs
Bacterial Gene Regulation I
Bacterial Gene Regulation II
Eukaryotic Gene Regulation I – Epigenetics and Imprinting (Erik Griffin)
Introduction to primary literature project (Natalia)
Eukaryotic Gene Regulation II
Eukaryotic Gene Regulation III
Exam # 4 – 7-10 PM
Project – group discussions
Project – group questions
Project – group discussions
Project – individual presentations
Mar 13
F
Final Exam (cumulative) – 8-11 AM
Lab Assignment Due Dates
Week of Feb 16 – Lab summary #1 due in your lab section
Week March 2 – In-class lab exam
Week of March 9 - Lab summary #2 due in your lab section
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Lab Introduction
The Bio13 laboratory will utilize the fruit fly Drosophila melanogaster to understand a system
used during development. The system centers around a protein found in all complex organisms
called Notch, which controls cell fate decisions. During the course of the term we will do the
following:
a) Look at the phenotypes of flies carrying mutations in the Notch gene and other genes in
the Notch system.
b) Look at the genetic interactions between different mutations in this system.
c) Determine the molecular nature of the mutations we are studying by PCR and DNA
sequence analysis.
d) Using the yeast-2-hybrid system, examine the interactions between proteins in this
system.
e) Examine the expression of genes downstream of Notch signaling using greenfluorescence protein assays.
This document contains general information about the lab, background on Notch signaling and
Drosophila genetics, important lab safety information, and a lab exercise on pipette operation
that you will be doing at the first lab meeting.
CONTENTS
General Information………………………………………………………………….. p2
Lab Schedule…………………………………………………………………………. p5
Background……………………………………………………………………………p6
Lab Safety……………………………………………………………………………p10
Pipette Operation……………………………………………………………………. p11
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General Information
Laboratory Instructors
Dr. Nicholas Sylvain
Office: LSC236
Telephone number: 646-8841
Web Site
We have set up a web site for the course on Canvas. The site will contain class lecture
notes, the lab manual and other useful items. The URL is: https://canvas.dartmouth.edu/
Lab Grade
The lab is worth 100 points which is 20% of the overall grade; it is broken down as follows:
Prelab and postlab assignments
Lab Summary #1
Lab Summary #2
50 points
25 points
25 points
Expectations of students prior to coming to the lab
All laboratory work can be done more effectively and efficiently if the subject matter is
understood before coming to the laboratory. To accomplish this, we expect that you will
read the background material and the experimental protocol before arriving in the
laboratory. Also, it may be helpful to read sections of the textbook that pertain to the
experiment being performed. The more prepared you are, the more efficient you will be in
successfully performing the experiments.
No one will be excused from the laboratory component of the class barring illness. Even so,
we cannot offer make-up labs for missed lab sessions for any reason including illness.
All laboratory experiments will begin with a brief discussion by the graduate TA of the
scientific basis for the experiment, the details of the experimental protocol, the location of
materials and other important information. Feel free to ask the graduate TA questions prior
to the lab (e.g. via email) or any of the lab staff (e.g. graduate TAs, lab instructor,
professors) during the lab.
Weekly lab assignments
Each week you will receive an assignment on Blackboard that will be due at the beginning
of your next lab section. The assignments must be submitted at the beginning of the lab
period. Post-lab assignments will be handed out at the beginning of lab, they must be
completed within the lab period and handed in to the graduate TAs before leaving lab. No
credit will be given for assignments that are handed in late. That means that you must come
to the lab on time and submit your post lab assignment before leaving the lab. The graduate
TAs will grade the assignments and hand them back to you during the next lab period.
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Lab Summaries
There will be two lab summaries submitted for this course:
Lab Summary #1 - Lab Summary #1 is due Friday, February 13 at the start of class.
Lab Summary #2 - Lab Summary #2 is due Friday, March 6 at the start of class.
The penalty for handing in lab summaries late will be minus 25% of the earned grade per
day that the summary is late.
Note: In order to pass the course, students must attend all labs and complete both lab
summaries.
Lab Partners
While performing the labs, you will work with a partner. The data generated in the labs
should be shared between lab partners. Although you are to work closely with your lab
partner while in the lab, the lab summaries and lab assignments are to be prepared
independently. Failure to write the lab summaries/assignments independently will be
considered a violation of the Dartmouth Honor Principle.
Academic Honor Principle
The Dartmouth College Student Handbook states “Fundamental to the principle of
independent learning are the requirements of honesty and integrity in the performance of
academic assignments, both in the classroom and outside. Dartmouth operates on the
principle of academic honor, without proctoring of examinations. Students who submit
work which is not their own or who commit other acts of academic dishonesty forfeit the
opportunity to continue at Dartmouth.”
There are a number of situations in which a student in Biology 13 might be unsure
about what constitutes a violation of the Academic Honor Principle. These situations
include (but are not limited to) the following:
a) Examinations must be completed without reference to written materials other than
those provided with the exam paper and must be completed without communication
with anyone else (the only permissible exception is that students may request
clarification of any exam question from the course faculty and staff who are present
expressly for that purpose). The answers that you provide must be entirely your own
work.
b) Under certain circumstances, we may allow exams to be re-graded by the instructors.
Any alteration of the answers between the time when the graded papers were
returned to the student and the time when the paper was submitted for re-grading
constitutes a breach of the Academic Honor Principle. To deter this practice, we
scan exam and assignment pages.
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c) Laboratory experiments are performed in pairs, and we encourage discussion and
collaborative analysis of the data. However, collaboration in organizing, outlining
or writing the lab summary is considered a violation of the Academic Honor
Principle. Any lab summary submitted for grading must represent the original
work (words, graphs, tables etc.) of the student submitting that lab summary. Do not
share computer files of work (including text, graphs, tables, etc.) to be submitted for
grading! The student misrepresenting the work of another as his or her own is in
violation of the Academic Honor Principle and it is quite possible that the Committee
on Standards might find the student providing the original file also to be in violation.
d) Weekly lab assignments must be prepared independently.
Honesty is the foundation of the academic pursuit of knowledge. In recognition of this, the
faculty and staff of Biology 13 will not overlook any violations of the Academic Honor
Principle. Indeed, the Faculty Handbook of Dartmouth College states explicitly that College
Faculty are obligated to report potential violations of the Academic Honor Principle to the
Dartmouth College Committee on Standards.
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Lab Schedule
January 13, 14 & 15
Lab #1:
Chromosomal DNA prep and PCR
January 20, 21 & 22
Lab #2:
Drosophila Genetics, setting up crosses
January 27, 28 & 29
Lab #3:
Gel electrophoresis, DNA sequencing
February 3, 4 & 5
Lab #4:
Drosophila Genetics, Scoring crosses
February 10, 11 & 12
Lab #5:
Analyze DNA sequencing data
Lab Summary #1 is due on Friday, February 13
at the beginning of lecture
February 17, 18 & 19
Lab #6
Set-up yeast matings
February 24, 25 & 26
Lab #7
Analyze yeast mating results
Drosophila transcriptional assays
March 3, 4 & 5
In-class lab exam
Lab Summary #2 is due at start of class on Friday, March 6.
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Background
Nearly all multicellular organisms begin with a single cell: a fertilized egg. This cell divides
numerous times to generate an adult animal. Therefore, this single cell eventually gives rise
to many diverse cell types such as muscle cells, brain cells, skin cells, and blood cells. A
key question in developmental biology is how a cell “decides” to become a certain cell type;
this is called a cell fate decision. The answer to this question is very complex and the field
of developmental biology has only begun to provide detailed answers. One signal that is
known to be involved in some cell fate decisions is Notch.
There are many proteins involved in Notch signaling, but for the purpose of this discussion
we will focus on 4 proteins.
Proteins involved in Notch signaling
1. Notch (N): The Notch protein is a receptor protein. The protein is expressed on the cell
surface and passes through the plasma membrane a single time. This is referred to as a
single-pass transmembrane protein. Therefore, a portion of the protein is found outside the
cell and a portion of the protein is found inside the cell. The Drosophila genome has a
single Notch gene; humans have four different Notch proteins.
2. Delta/Serrate: The Notch protein is activated by a ligand on an adjacent cell. This ligand
is also a single-pass transmembrane protein. To activate Notch the extracellular portion of
Notch interacts with the extracellular portion of the ligand. In Drosophila there are two
Notch ligands: Delta and Serrate. In humans there are five Notch ligands: Delta-like1,
Delta-like2, Delta-like3, Jagged1 and Jagged 2.
3. Suppressor of Hairless (Su(H)): Su(H) is a transcription factor that binds to the 5’
noncoding region of specific genes. In the presence of the Notch signal the transcription of
these specific genes is activated. In the absence of the Notch signal the transcription of
these specific genes is not active due to the presence of a repressor complex that binds to
Su(H). In humans, the Su(H) protein is called CBF1; in worms it is called lag-1. It is not
uncommon for the protein to be called CSL (CBF1, Su(H), Lag1).
4. Hairless (H): A complex of proteins binds to Su(H) in the unstimulated state. One of
these repressor proteins is called Hairless. In humans there is also a repressor complex, but
there are no proteins that are clearly similar to Hairless.
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How do these proteins
produce a signal?
A simplified version of Notch
signaling is shown in the figure
on the right. When the
extracellular portion of Notch
comes in contact with the
extracellular portion of the
ligand, two proteases cleave
Notch at the membrane. This
releases the intracellular portion
of the Notch protein. The Notch
intracellular domain migrates
into the nucleus and, along with
other proteins, turns on the
transcription of target genes by
binding to and activating
Su(H)/CBF1. In the absence of
the Notch intracellular domain,
transcription of these same
genes is shut off due to the
interaction of Su(H)/CBF1 and a
complex of repressor proteins.
The final output of this signal is
the transcriptional induction of
specific genes. All of these
genes have a Su(H) binding site
in the 5’ noncoding region.
Genes that are activated by
Notch include but are not
limited to: enhancer of split,
simple-minded, veinlet, and
twist.
Notch Signaling: Notch is activated by binding Delta on an adjacent
cell. This induces two proteases to cleave Notch at the membrane. The
intracellular domain of Notch (Nintra) translocates into the nucleus and
binds CSL displacing the repressor complex (Co-R). Nintra, CSL, and
other proteins (Co-A) act as transcriptional activators of a subset of
genes. In Drosophila, CSL is Su(H), and Hairless is part of the Co-R
complex. Lai, E.C., (2004) Dev. 131: 965-973
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Roles of Notch Signaling in Development
a. Restricting cell fates (inhibitory Notch signaling)
Imagine a group of cells that all have the potential to become neurons. One of these cells
expresses Serrate on its cell surface. The Serrate protein will then interact with Notch on
adjacent cells. The Notch signal inhibits
the default neuronal cell fate and the cells
instead become endothelial cells.
This results in a single neuronal cell
surrounded by non-neuronal cells.
A
B
C
The best-studied case in Drosophila
Dolph, P.J., 2007
involves the sensory organ precursor cells
All 7 cells in “A” have the potential to adopt the
(SOPs). SOPs start as undifferentiated
same cell fate. In “B” one of the cells is selected
and expresses a Notch ligand on the surface. This
cells. In the adult, one cell is selected to
turns on Notch signaling and these peripheral cells
become a neuron (the cell connected to
change their cell fate. This results in one cell of one
the bristle) and the other cells become
type surrounded by cells of a different type (C).
non-neuronal cells, such as sheath cells
and socket cells through inhibitory Notch
signaling.
b. Specifying cell fate and creating boundaries. (inductive Notch signaling)
Imagine two different tissue types that meet and form a boundary. Delta or Serrate are
expressed on the surface of these two tissues and signals to the boundary cells. When Notch
is turned on in the boundary cells, the transcriptional signals specify a new cell fate. In
Drosophila, the wing margin is made by inductive signaling. This is why in Notch mutants
there is a disruption in the wing margin and notches appear in the wing.
v v v v
v v v v v v
A
v v v v
v v v v v v
B
v v v v
v v v v v v
C
Dolph, P.J., 2007
In “A” there is a boundary formed between the gray cells and the striped cells. Notch ligands
in those cells turns on Notch signaling in the white boundary cells (B). This induces them to
change their cell fate and a new cell type is formed at this boundary (C).
During the course of the term we will analyze the Notch/Su(H)/Hairless system from a
genetic and molecular perspective. The lab will focus on 4 aspects of this system.
1. The genetic interactions between Notch, Su(H), and Hairless
2. The molecular basis of the mutations found in Notch, Su(H), and Hairless alleles
3. The molecular basis of the phenotypes seen in Notch, Su(H), and Hairless alleles
4. Interactions between the Notch, Su(H), and Hairless proteins.
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Drosophila Genetics
The fruit fly Drosophila melanogaster has been used extensively as a model organism to
study biological problems. There are many reasons why Drosophila has been used:
1. Its small size enables the researcher to grow large numbers of flies in a small space.
2. It has a very short generation time (about 2 weeks at room temperature).
3. It has high productivity with a single female capable of laying over 500 eggs in a week’s
time.
4. Over 100 years of research has resulted in thousands of genetic mutants and a large
number of genetic tools.
5. The animal is easily transformed (genetic material can be introduced and stably
integrated into the genome)
6. Full sequenced genome.
Life Cycle
The life cycle of Drosophila lasts for about two weeks at room temperature. Inseminated
females lay eggs on the surface of the food. Larvae hatch from the eggs within 24 hours.
The larvae progress through three larval stages (called 1st, 2nd, and 3rd instars) by successive
moltings. The 1st and 2nd instar stages last approximately one day each and the 3rd instar
stage lasts about 2 days. Late in the 3rd instar stage the larvae crawl on the side of the glass
searching for a place to pupate. The pupal stage lasts 4-5 days during which the body is
completely remodeled. The adult then emerges from the pupal case.
Females are not receptive to mating until about 12 hours of age. Courtship between males
and females follows a stereotypical series of events that includes a song that the male
performs by vibrating one of his wings. Once inseminated, females can lay over 100 eggs
per day.
It is very easy to distinguish between male and female fruit flies. Males are smaller than
females and have a darker and more rounded abdomen. Males also have a small comb-like
structure on their front legs called a sex comb; it is used to grasp females during mating.
The Drosophila genome
Drosophila has three pairs of autosomal chromosomes and an X and a Y. The 2nd and 3rd
chromosomes are metacentric and contain more than ¾ of the genetic material in the fly.
The 4th chromosome is very small. Females have two X chromosomes and males have one
X and one Y. The X chromosome is telocentric and is about ½ the size of the 2nd or 3rd
chromosomes. As in humans, the Y chromosome has a limited amount of coding
information. The size of the genome is about 165 million base pairs and contains an
estimated 14,000 genes.
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Orientation to the Laboratory: Rules of Conduct and General
Safety
1.
Place all jackets, unnecessary books, purses, backpacks, and paraphernalia on the hooks
and cubbies in the vestibule to the lab. This includes cell phones and personal listening
devices. Cell phones should be turned off for the duration of the lab. The laboratory
work area must be kept free of articles not actually in use.
2.
Eating, drinking, and smoking are forbidden at all times in the laboratory.
3.
Long pants and close-toed shoes must be worn in the laboratory.
4.
Do not place anything in your mouth while in the laboratory. This includes pencils,
food, and fingers. Learn to keep your hands away from your eyes, nose, mouth, etc.
5.
Know the location of safety equipment (fire extinguisher, eyewash, emergency shower,
and first-aid kit).
6.
Know the location of the nearest fire exit.
7.
Return all reagents, cultures, and glassware to their proper places.
8.
Avoid contamination of the benches, floor, and wastebaskets.
9.
Clean your work area (laboratory bench) with a disinfectant (70% ethanol) before and
after each laboratory period. This standard procedure lessens the chance for accidental
infection as well as for contamination of lab materials.
10. Wash your hands thoroughly before and after each experiment, using soap. Always
wash your hands before leaving the laboratory.
11. You should avoid wearing loose clothing and long hair should be tied back to minimize
fire hazard and contamination of experiments and cultures.
12. Never take laboratory materials outside of the laboratory workspace.
Sterile Technique
1. Wash your hands before starting your experiment and throughout the lab period.
Always wash your hands before leaving the lab and when you return to the lab.
2. Clean the bench area with 70% ethanol before you begin the lab exercises and when you
have finished for the day.
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3. Do not touch sterile surfaces or sterile solutions with non-sterile surfaces (such as your
fingers).
4. Keep sterile containers covered when not in use. This includes Petri dishes, sterile
pipette tip containers and bottles or tubes containing sterile liquids.
5. Do not reuse pipette tips. Use a new, sterile tip for each transfer of liquid.
Pipettor Operation:
Pipettors are precision instruments and very expensive. Be sure to place them on the lab
bench in a secure place when not in use. Do not drop them!
You will use pipettors in most of the lab exercises this term. The successful completion of
your lab work will require the proper use of pipettors.
When using pipettors, be sure that you have the correct model for the volume you want to
measure. You will work with three different pipettor models this term, P-20, P-200, and P1000. Each model is identified by a number on the top of the plunger. For example, the P20 has "20" on the top of the plunger.
Each pipettor model has a defined volume range. They are as follows:
Pipette Model
P-20
P-200
P-1000
Volume Range
2-20 µl
20-200 µl
200-1000 µl
Tip Color
Clear
Clear or Yellow
Clear or Blue
1.
Set the desired volume on the digital volume indicator by holding the pipettor body in
one hand and turning the "volume adjustment knob" until the correct volume is
displayed.
2.
Attach a disposable tip (these tips are sterile, so be careful not to contaminate them) to
the shaft of the pipettor. Press it firmly with a slight twisting motion to insure a strong,
air-tight seal.
3.
Using your thumb, depress the plunger to the FIRST STOP POSITION. This part of
the stroke is the calibrated volume displayed on the volume indicator.
4.
Holding the pipettor vertically, immerse the tip into the sample liquid.
5.
With your thumb positioned on the plunger, allow the plunger to SLOWLY return to
the "up" position. Never permit it to snap up quickly!
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6.
Wait 1-2 seconds to ensure that a full volume of sample is drawn into the tip. Viscous
fluids in particular will require additional time to flow into yellow or clear pipette tips,
which have a small orifice.
7.
Withdraw the pipettor tip from the solution. If any liquid remains on the outside of the
tip, touch the tip to the interior wall of the tube to remove the excess.
8.
Look at the volume of liquid in the pipettor tip. Does the tip appear to contain the
expected amount of liquid (we will teach you how to determine this)? If so, proceed to
step 9. If it does not, please check with the TA before proceeding to step 9.
9.
To dispense the sample, depress the plunger slowly to the FIRST STOP. Wait 1
second. Then depress the plunger to the SECOND STOP POSITION (bottom limit of
stroke), expelling any residual liquid in the tip.
10. With the plunger still fully depressed, carefully withdraw the pipettor.
11. Return the plunger slowly to the "up" position.
12. Discard the tip into the used tip beaker provided by sharply depressing the "tip ejector
button"
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