biology 13 gene expression and inheritance · biology 13 syllabus winter 2015 3 and can result in a...

Biology 13 SYLLABUS Winter 2015

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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-slash-advisory-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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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 10% Second Exam 10% Third Exam 15% Fourth Exam 15% Final Exam 20% Project 5% Participation (pre-lecture and in class) 5% Laboratory 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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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 M Course overview Jan 7 W DNA as Genetic Material, DNA Structure Jan 8 Th Course study tips (led by Natalia) Jan 9 F Genomes/Chromosomes/Chromatin Jan 12 M DNA Replication Jan 14 W Transcription I Jan 15 Th Transcription II Jan 16 F Exam #1 (in class) Jan 19 M Martin Luther King Day – no class Jan 21 W Transcription III Jan 22 Th Genetic Code Jan 23 F Protein Synthesis - Translation Jan 26 M Mutation Jan 28 W Effects of Mutation Jan 29 Th Genes and Gene Products Jan 30 F Exam #2 (in class) Feb 2 M Meiosis Feb 4 W Patterns of Inheritance I – dihybrid cross Feb 5 Th Patterns of Inheritance II – deviations Feb 6 F Winter Carnival – no class Feb 9 M Patterns of Inheritance II – sex linkage Feb 11 W Linkage and Mapping I Feb 12 Th Linkage and Mapping II Feb 13 F Pedigrees Feb 16 M In class review

Exam #3 – 7-10 PM Feb 18 W Mapping with Molecular Markers Feb 19 Th GWAS, and QTLs Feb 20 F Bacterial Gene Regulation I Feb 23 M Bacterial Gene Regulation II Feb 25 W Eukaryotic Gene Regulation I – Epigenetics and Imprinting (Erik Griffin) Feb 26 Th Introduction to primary literature project (Natalia) Feb 27 F Eukaryotic Gene Regulation II Mar 2 M Eukaryotic Gene Regulation III

Exam # 4 – 7-10 PM Mar 4 W Project – group discussions Mar 5 Th Project – group questions Mar 6 F Project – group discussions Mar 9 M 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

BIO13 WINTER 2015 INTRODUCTORY MATERIALS

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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 green-

fluorescence 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 50 points Lab Summary #1 25 points Lab Summary #2 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. The best-studied case in Drosophila involves the sensory organ precursor cells (SOPs). SOPs start as undifferentiated cells. In the adult, one cell is selected to become a neuron (the cell connected to the bristle) and the other cells become 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.

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.

A B C All 7 cells in “A” have the potential to adopt the same cell fate. In “B” one of the cells is selected and expresses a Notch ligand on the surface. This turns on Notch signaling and these peripheral cells change their cell fate. This results in one cell of one type surrounded by cells of a different type (C).

Dolph, P.J., 2007

A B C 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).

Dolph, P.J., 2007

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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 P-1000. Each model is identified by a number on the top of the plunger. For example, the P-20 has "20" on the top of the plunger. Each pipettor model has a defined volume range. They are as follows:

Pipette Model Volume Range Tip Color P-20 2-20 µl Clear P-200 20-200 µl Clear or Yellow P-1000 200-1000 µl 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"

biology 13 gene expression and inheritance · biology 13 syllabus winter 2015 3 and can result in a...

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