Aparna Sreenivasan

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Biology 361L Eukaryotic Molecular Biology Lab

Fall 2011

Instructor: Dr. Aparna Sreenivasan
Office: Building 53, E207
Lab: Building 53, E325
Phone: 582-3210
Office hours: Mondays 11-12 and by appointment.
E-mail: aparna_sreenivasan@csumb.edu


Lab manager: Danitza Alvizar
E-mail: dalvizar@csumb.edu
Office: Bldg 53 3rd floor

Readings: We will read journal articles, news stories and other selections during the course. There is currently NO text to purchase.

Description:

This molecular biology laboratory course, which uses a project approach to learning and incorporates an independent research component, was designed to enhance the preparation of students for careers in research, biotechnology and science education, increase knowledge retention and integration of concepts among biology majors and teach students what it is like to work in a laboratory setting.

In this laboratory you will carry out a series of experiments employing both routine and advanced techniques in molecular biology. The first set of laboratory exercises is designed to simulate a project that might be carried out in an actual research laboratory at an R1 institution. You will learn how to propagate and utilize the Saccharomyces cerevisiae, bakers yeast, as a model organism and apply a complex technique, called microarray analysis to look at whole genome expression in specific conditions in the laboratory. In addition, you will take real sequencing data from the Drosophila dot chromosome sequencing project and annotate Drosophila sequences for Washington University in St. Louis. The second project could culminate into a scientific publication with Washington University Researchers and other students around the country who are also helping to annotate the Dot Chromosome.

Just like "real world" laboratories, we will have days where we focus on studying what we are doing between our major projects, through journal clubs, smaller experiments, class discussion (ie. Lab meeting) and database analysis. Some of the smaller experiments include preparatory bioinformatics workshops. As this lab has been designed to teach you how to work in an industrial or academic research setting, you may sometimes be expected to come and "check your organisms" outside of class time, and all of your experiments may not work. It is up to you to take copious notes and be able to trouble shoot your experimental protocols and reflect on what you may do differently the next time around. You will notice that your laboratory notebook is worth 15 percent of your grade, this book is not intended to be a slapped together bunch of pages, but an actual lab notebook, like one that you would expect to keep if you were working in biotechnology or in graduate school.

You will also work on an independent research problem that uses the same system and tools and will present your research on that problem to the class at the end of the semester. As you will also note, this course has been designed to speak to many types of learning styles, your assignments range from presentations, to write ups and worksheets to a final exam. So each person’s strengths should be represented by one of these formats.

The prerequisites for BIO 361L are:

Biology 241/241L: Introduction to Cell and Molecular biology with lab. Biology 320: Microbiology = recommended Biology 311: Genetics = Recommended corequisite

BIO 361L fulfills part of MLO10 for Biology majors. This course is also an approved UD elective course for Biology and ESTP (with advisor consent).

The Outcomes for BIO 361L are:

During the course students will:

  1. apply knowledge from introductory cell and molecular biology and genetics to answer key scientific questions in the laboratory.
  2. become proficient in the propagation and utilization of the model organisms S.cerevisiae and (in some semesters) C. elegans for use in molecular biology research
  3. read and present scientific papers in journal club format.
  4. develop and maintain a laboratory notebook with a distinct protocol section.
  5. analyze large-scale genomic data.
  6. use real world computer programs and functions to annotate and finish genome projects.
  7. contribute to scientific literature through a coordinated project with Washington University in St. Louis.
  8. learn how to independently identify molecular biology projects that funding agencies will support.

By the end of the course students should be able to:

  1. determine what is known about an area of research by exploring the published data.
  2. pose a scientific question and develop a testable hypothesis relating to a gap in knowledge.
  3. design experiments with appropriate controls to answer the question or test the hypothesis.
  4. carry out experiments using laboratory techniques covered in the class.
  5. explain the principles underlying those techniques.
  6. collect, analyze and interpret experimental data, and
  7. communicate your results to others.

WHAT YOU CAN EXPECT FROM ME:

I will come to each class session prepared to facilitate a productive class period. I have done my best to select interesting, well-written material for you to read and write about, and that I will do my best to make good use of class time. I promise you in-depth feedback on your written work within two weeks of you turning in an assignment. I will be available to meet with you during my office hours or at whatever other times we arrange. I will take your concerns and interests extremely seriously. If you don’t like the way I have structured class time or feel that I have assessed you unfairly, please speak with me or email me right away. I always respect honest feedback; it is my job to help you get as much as possible out of the class.

Texts:

There are no required texts. The following books may be useful, depending on your background.

These books will be in my office for perusal.

  • "Bioinformatics and Functional Genomics" by R. Pevsner, 2009 (2nd ed.), J. Wiley & Sons, NJ (recommended for Bio majors if you would like more introduction to the computer tools we use).
  • "BLAST" by I. Korf, M. Yandell, J. Bedell, 2003, O’Reilly (ISBN 0596002998) (recommended for in-depth use of BLAST and interpretation of results).
  • "Genomes 3" by Terence A. Brown, 3rd ed, 2006, Garland Science (ISBN 0815341385)

(recommended if no equivalent book, such as the texts for Bio 2970, are in hand).

Capstone projects:

This course is an approved Biology capstone project course, students that wish to generate capstone projects from their course work should approach the instructor by week four of the semester. You must pass the BIO361L course to continue with your capstone project. The capstone project will be a GROUP capstone with 2 other individuals from the class.

Lab partners: For most of the course, you will work with lab partners. But you may not keep the same lab partners all semester. In this course, we sometimes change lab partners. Why? The problem with permanent pairings is you tend to get stuck in a rut, always doing the same portion of the work (Josiah always pipettes while Jamie always does the math, for instance), and never trying (or letting the other person try) something truly challenging. When you change lab partners, you break these patterns and gain the opportunity to learn new skills.

I also want to emphasize the collaboration skills you gain by engaging with new co-workers. Whether you go on to biotech, medicine or academia, you will continually work with new and varied people. In fact, the most effective collaborations emerge when people with different backgrounds and expertise put their heads together and learn from each other. So maybe you won’t like the change-ups at first, but I hope someday you will see why they are valuable.

Flowsheets: In preparation for each laboratory exercise, you should produce a labeled diagram of the day’s work in your notebook. This flow sheet is to be based on the readings and protocols distributed on iLearn. Only the procedures to be performed on any given day need be included. In making your flow sheets, avoid copying the steps from the protocol. Instead, make a diagram or series of pictures: draw tubes and pipets, use arrows to connect steps or to indicate the transfer of liquids. Be sure to include necessary experimental details, such as times and temperatures. Flow sheets will be checked at the beginning of class on certain days (listed separately on iLearn). They will not be accepted after class has begun.

Module quizzes: One concept quiz will be given at the end of each module, normally on the first day of the next module. These quizzes are worth 10 points and will usually involve written answers rather than multiple choice. When quizzes are administered through iLearn, they must be completed in the assigned time frame. No make-up quizzes are given.

Reading Quizzes. These are given at the beginning of a class period to ensure that everyone comes prepared for the day’s work or discussion. Reading quizzes are based on reading assignments given in class. Quiz times will be random, so you should always come prepared. There will be three reading quizzes throughout the semester.

Personal Devices: Do not send text messages, check email, or play Nintendo during lecture and discussion. I get a thrill out of calling on people who appear distracted, so keep your head in the game. Also, please silence cell phones, BlackBerries, walkie-talkies, and the like. And please don’t listen to your iPod during class. I also request that you do not record my lectures and our discussions. We have four hours in lab (8 total per week) and I want you to be engaged. So come ready to participate and be a part of a lab group.

Required Supplies: You will need to purchase some supplies or borrow them from previous BIO 361L students; some can be shared. You can find most of these items at the student bookstore and at most drugstores.

  • Lab notebook (bound, pre-paginated, 8.5 X 11" style only)
  • Flash drive (100 MB or more) for taking computer work and data home

Department Computers:

Computers will be required for a number of classes. You are welcome to bring your own and use it during these periods, provided it is capable of running the necessary software. On days when computers are needed, I will provide computers with wireless internet and printer access for you to sign out if you prefer. When you put these computers away, please follow these steps.

  1. Throw away any temporary materials and empty the trash.
  2. From the Apple menu in the upper left, select Shutdown. Click OK.
  3. Wait for the screen to go blank before closing the computer. Otherwise it will merely sleep in a partly shut down state.
  4. Return the computer and adapter to the correct shelf on the cart.

Attendance:

Attendance is mandatory: You cannot miss labs. Your final grade will be docked by two percent for every lab session that you miss. The reason for this harsh policy is that by missing lab class, you are putting all of the work on your partner and relying on them to provide you with what you should be doing together. That is unfair. In addition, we cannot have make up labs, it takes a lot of prep to set up each lab session and we cannot backtrack.

Late Policy:

I do not accept late work. In the real world, if I turn in a grant application even one hour late, the funding agency will automatically reject it. That said, if extenuating circumstances should arise that preclude you from turning something in, you must call me, or contact me immediately and let me know the situation. I will take these situations on a case-by-case basis.

Academic Honesty Summary Statement:

"Within the SMART College, plagiarism and other forms of academic dishonesty are not tolerated. Students who are found by faculty to have committed plagiarism in an assignment will, at minimum, upon the first offense fail that assignment. Upon the second offense, a student will, at minimum, fail the class. Each incident and the student's name will be reported to the college administration. (SMART Academic Honesty Website address)"

I take plagiarism very seriously, we will go over the parameters of plagiarism and academic honesty carefully in class so that everyone is clear on the issue.

A NOTE FOR STUDENTS WITH DISABILITIES

Those who may need specific accommodations please see me as soon as possible during office hours or make an appointment to see me. Also contact: Student_Disability_Resources@csumb.edu Phone: 831-582-3672 voice, or 582-4024 fax/TTY; http://sdr.csumb.edu/

Tentative schedule:

A current copy of this syllabus and all necessary course documents can be found on ilearn under BIO 361L.

Day Date Description Deliverable
Tuesday August 23
  • Introduction to the lab
  • How to read a scientific paper.
  • Discussion Scientific Questions.
  • Lecture: Emerging technologies
None
Thursday August 25
  • Observe yeast, streak plates
  • Introduction to the yeast genome database, pubmed.
  • Begin gene of interest projects
Module Quiz on how to read a scientific paper from Tuesday
Tuesday August 30
  • Lecture: Introduction to microarrays:
  • Journal club 1
  • Groups 1 and 2: DeRisi Diauxic Shift paper and PLOS paper
Your gene of choice to Aparna
Thursday September 1
  • Introduction to microarray experiment
  • Discussion and calculations for growing yeast cultures, set up harvest schedules.
Tuesday September 6
  • Grow yeast cultures, treat in conditions
  • Harvest yeast, spin down, freeze (on own time) TBD
Flowsheet 1: Yeast growth and harvesting (all groups do all conditions on flowsheet)
Show Aparna the papers that you gathered about your gene of choice
Wednesday September 7 Harvest yeast continued.
Thursday September 8 RNA isolation and quantification by gel electrophoresis Flowsheet 2: RNA isolation and quantification by gel electrophoresis
Tuesday September 13
  • RNA Isolation and quantification by gel electrophoresis
  • While gels are running do gene of interest presentations.
Thursday September 15 Convert RNA to cDNA Flowsheet 4: reverse transcription
Tuesday September 20
  • Gene of interest project: Primer design exercise and design primers
  • Order primers
  • Practice Data Analysis introduce magice tool
Flowsheet 5: Primer design
Thursday September 22
  • Practice DATA analysis. Magic tool.
  • Download magic tool, do magic tool Raven data analysis.
  • Analyze De Risi Godlist.
Tuesday September 27
  • Hybe arrays
  • WEDS: Wash and hybe with dendrimers, wash again. Store in dark until Thursday.
Flowsheet 6: methods of data analysis with genepix
*remember to work on your gene of interest project
Thursday September 29 Read arrays at Hopkins None
Tuesday October 4 Work day
Thursday October 6
  • Journal Club 2: Advancements with microarray analysis
  • Analyze microarray Data
  • Groups 3 and 4
Tuesday October 11
  • Shift to Genomics: The Research problem Intro Lecture:
  • Overview of DNA sequencing (goals) the pipeline and strategies. Lab: Intro to laptops, intro to Unix, intro to BLAST, scripted walk through Lecture: The C value paradox, Intro to BLAST
  • Watch virtual tour and next generation technologies.
Passed out initial drosophila dot chromosome paper and sample annotation project.
Thursday October 13
  • Lecture: Chromatin structure and heterochromatin
  • Lab:
  • Lecture: Gene finding: detecting and interpreting genetic homology.
  • Lab: Simple annotation exercise.
Peer review Microarray writeups
GOI: PCR with primers.
Tuesday October 18
  • Lecture: The drosophila dot chromosome: and discuss DOT paper.
  • Lab: Detecting and Interpreting Genetic homology CW1
  • GOI: Run PCR on gel
  • Microarray writeup due
  • Send GOI PCRs for sequencing
Thursday October 20 Lecture: Introduction to gene predictors. Efficient gene finding in D. grimshawi. GOI: Transform PCR into yeast
Tuesday October 25
  • GOI project: Analyze sequences. Present sequences to class. Transform PCR product into yeast strain to induce homologous recombination.
  • Journal club 3: Genomics and sequencing papers Groups 5 and 6
  • Module quiz 2
  • GOI: Check yeast for growth – assess phenotypes
  • CS HW1 due
Thursday October 27 Lab: Introduction to multiple sequence alignments; use of Clustal. Lab: Clustal analysis of one feature (translated portion and regulatory element). GOI: restreak yeast
Tuesday November 1 Look at plates to see if any visible phenotypes arise. Streak positives, freeze down in 3 days.
    • remember to work on your gene of interest project
  • CS HW 2 due
  • GOI : dirty PCR
Thursday November 3
  • Lecture: Design and use of repeatmasker; other ways to find repeats.
  • Lab: continue analysis and annotation of own fosmid, particularly repeats.
None
GOI: Run dirty PCR on gel
Tuesday November 8
  • Lecture: Finding repeats in Drosophila
  • Lab: Continue analysis and annotation of own fosmid.
Thursday November 10
  • Continue to analyze and annotate own fosmid.
  • Presentation of results from GOI project.
Presentation of results from GOI project.
Tuesday November 15 Veteran’s day no lab None
Thursday November 17
  • Lecture: How to prepare annotation reports.
  • Lab: Preparation of annotation reports, synteny.
None
Tuesday November 22 Fall break no lab *remember to work on your gene of interest project
Thursday November 24 Fall break no lab None
Thursday November 29 Fall break no lab None
Tuesday December 1 Final annotation reports: your fosmid, with a map of genes, repetitious elements, and alignment to melanogaster, including a Clustal analysis. Complete annotation of all exons; include a map and discussion of synteny. As time permits, investigation of the nature of the repetitious elements; search for noncoding conserved regions; search for endpoints of genes; search for regulatory elements; etc. Follow‐up session on course evaluation
Thursday December 6 Annotation presentations 1-9 Presentation
Tuesday December 8 Annotation presentations 10-18 Presentation
Thursday December 13 Lab final Final exam
Tuesday / Thursday December 15 Gene of Interest writeup and annotation report due. None

Grading:

Assessment will be based on the following items:

Parameter Points Percentage
Laboratory notebook 50 11
Journal Club
40 points for presentation, 10 points for questions.
50 11
Flowsheets 25 5.5
HW 1-2 Genomics 50 11
Gene of Interest study 50 11
Gene of interest presentation 25 5.5
Annotation report 50 11
Microarray data analysis 50 11
Annotation presentation 25 5.5
Module quizzes 25 5.5
Final exam 50 11
Total 450 100

Grading will be on a straight scale:

A+ 99-100
A 94-98
A- 90-93.99
B+ 87-89.99
B 84-86.99
B- 80-83.99
C+ 77-79.99
C 74-76.99
C- 70-73.99
D+ 67-69.99
D 64-66.99
D- 60-63.99
F below 60

There is no extra credit for this class.

Laboratory Notebooks:

Experiments are pointless unless you write down what you do. Your lab notebook is your record of every experiment you do, from conception to conclusion. In it you need to state the question your experiment is intended to answer, describe the experimental approach, and give the needed protocols. More importantly you keep a real-time account of how the procedures go, observations, changes, errors, and questions that arise as you carry out the experiment. To the extent possible, all data is recorded in the notebook. You also write the location of any reagents you store, along with how the tubes are labeled and what they contain. If you do this right, you, or anyone else, should be able to go back to any experiment you have ever done, find out what you did, what the results were, repeat the experiment exactly, or find any of the reagents or computer data you stored.

For this class, you will need to purchase a bound, pre-paginated, laboratory notebook. Essay style notebooks are not acceptable and will not be scored. You may use a partly-full notebook from another class, as long as it is of the approved type.

Notebook Scores:

On five days notebooks will be checked in class and scored for specific features. Each check day is worth 10 points. These days will not be announced until the day of the check. Some checks will be made by the instructor. Other times you will complete a worksheet that asks for specific details from an earlier experiment. So it is essential for you to make entries in your notebook as you work.

Table of contents

Reserve the first page of the notebook for a running TOC. Add experiments and their corresponding page numbers to the table throughout the semester.

Top of page

The top of each page in your notebook must include a page number, the date the page was written on, your name, and the name of the experiment.

General format

Do not leave blank pages. If you cross out an entry, use a single line so that the entry can be read. If you change a primary datum, write a brief explanation nearby. You do not need to explain a change in a calculation or in prose sections like the purpose and conclusion.

Objective

For each lab module (not each day), write an overall objective. State the question or hypothesis the experiment will answer or test, and give the approach (how you will answer the question). Often the approach can be phrase as a prediction, such as, "if we do A, then B will happen." Reference any paper or resource from which procedures were taken.

Procedure

You do not need to copy the procedures from the materials I provide. That is just busy work. You aren’t training to be a medieval scribe. Instead, write down the title of the provided procedure and include a printout of the procedure in the back of the notebook. Also spell out what the procedure achieves. For instance, you could write "isolated total RNA from the yeast harvested on [date] using procedure ‘RNA isolation.’" It is not okay to write "did procedures for today." Imagine reading that in 6 months and trying to figure out what you did that day.

It is also very important to keep notes of any changes to the protocol, errors made, details not in the protocol, and specific observations you make as you go (see next section). For instance, if the procedure says, "at this point you should have 5 to 10 l in the tube," write down the actual volume you have (e.g. "after purifying my RNA, I had a final volume of 7 l"). You must also write down the results of any measurements taken during the procedure ("by Nanodrop spectroscopy of 2 l of sample, the RNA concentration was 900 ng/l, 260/280 ratio was 2.1")

Observations

The whole point of a lab notebook is to make up for the weakness of human memory. The only way it can hold an accurate record of what you did in the lab is if you write down observations right away as you make them. Your notebook should not be a diary written at the end of the day or a memoir compiled weeks later. Only an accurate record will allow you and others to reproduce your work and to find and understand the precious reagents you generate in the laboratory for use in future experiments.

Therefore, always record the data from each lab exercise as you collect it, and write your observations as you make them. Do not write on scraps of paper with the intention of transferring the information to the notebook later. Always write directly in your notebook. Here are some examples of what to do in this section of your experimental record.

  • Keep a running commentary of observations, particularly when you weren’t sure what to expect. "The cell lysate was viscous and hard to pipette." "A few small air bubbles were trapped under the coverslip." "The supernatant was cloudy."
  • Draw and describe microscope observations. Include measurements of features.
  • Build tables to record repeated measurements.
  • If your data include printouts, photographs, dried gels or the like, label these thoroughly

and tape them into the notebook near where they are described.

  • Stored or frozen samples must be labeled with the contents, the date, the student (or

group) name, and this entire label should be recorded in your notebook, along with the storage location.

  • Record ideas and questions that arise during the procedure.
  • Record any mistakes or deviations from the protocol as soon as they happen, how they may affect the outcome of the experiment. and any steps you take to adjust for the error.
  • Conclusions. In several sentences or bullet points, state the results of the experiment as simply and directly as possible. Draw appropriate conclusions. Mention any caveats. Draw or tape in any graphs or tables needed. The conclusions should be written no later than the first day of the next module.

Laboratory procedures

  1. All workspace (including common-use areas) must be cleared of all glassware, equipment, and reagents at the end of each laboratory period.
  2. Do not write directly on any glassware or equipment with any type of lab marker. Instead, use label tape for all labeling. Remove the tape before returning the equipment or putting the glassware into the wash.
  3. Leave dirty glassware on the cart near the front of the room, unless otherwise instructed.
  4. Place re-usable glass pipets in the flat plastic boxes provided.
  5. Dispose of needles and razor blades in the sharps container provided.
  6. Put broken glass in the cardboard box specially marked for that purpose.
  7. Throw old petri dishes and other biologicals in the biohazard kick-buckets.
  8. Most chemical waste must be placed in the labeled containers for disposal, not poured down the sink. If you aren’t sure, ask.
  9. Please place ordinary trash — paper towels, wrappers, etc. — in wastebaskets, rather than in the biohazard waste.
  10. When you have finished cleaning up, wipe the tabletop at your station down with 70% ethanol.

Safety

  1. Please listen closely to instructions about the handling of hazardous chemicals.
  2. Wear goggles and latex gloves when handling corrosive solutions.
  3. Be sure to wear clothing and shoes that maximize protection.
  4. Smoking is not permitted in any indoor areas on campus.
  5. Wash hands before leaving the lab and before eating.
  6. No food or drink is allowed in the lab.
  7. Tie back long hair when working near flames or entangling equipment.
  8. Never mouth pipette. Pipette pumps are provided.
  9. Always report spills or accidents to the instructor or T.A. immediately.
  10. Know the location of the emergency eyewash, shower and fire extinguisher.