PCB 3063L General Genetics Laboratory Course Syllabus
Course Supervisor: Dr James Garey (974-3900) garey@chuma1.cas.usf.edu
Teaching assistants:
Jamie Colson (SCA 224) jcolson@brill.acomp.usf.edu
Maren Jensen (SCA 224) mjensen@mail.usf.edu
Thomas Han (SCA 224) han@helios.acomp.usf.edu
Prerequisites: Students must have completed or be currently enrolled in the General Genetics lecture course (PCB 3063). Students who drop the lecture section of the course will not be allowed to continue in the laboratory section without departmental permission.
Laboratory Materials:
Laboratory Manual (Download the PDF file from: http://chuma.cas.usf.edu/~garey/labmanfall00.pdf and print it yourself – it is best to do this in a computer lab (e.g. SCA 222) where you can use the laser printer which is more permanent than inkjet printers. Cost of printing in the computer lab is approximately 5¢ per page and the manual is 59 pages long.
Safety Glasses
Colored Pencils, Sharpies (fine point)
Laboratory Notebook (Quad-ruled composition book)
Course Objectives: The course is designed to complement and reinforce the principles taught in PCB 3063. The laboratories are designed to illustrate fundamental genetic concepts and students will learn to collect, analyze and present experimental data in formal laboratory notebooks and scientific laboratory reports.
Grades: Final grades are assigned according to the following scale:
A 89.5 – 100
B 79.5 – 89.4
C 69.5 – 79.4
D 59.5 – 69.4
F < 59.5 %
All posted grades are final unless they are the result of a miscalculation. Late drops, changes to the S/U grading system or incompletes will not be granted except under those circumstances specified in the USF undergraduate catalogue. No exceptions. Plus/minus grading will not be used.
Attendance: Attendance is mandatory. Please turn off cell phones during class. Students who anticipate the necessity of being absent from class due to the observation of a major religious observance must provide notice of the date(s) their TA, in writing, by the second week of classes. Students with disabilities must provide written notice of any special consideration to their TA by the second week of classes. Un-excused absences will result in a 10% reduction in your final grade. In general, verifiable absences are dealt with on a case by case basis and are seldom excusable. You may only attend the laboratory section for which you are registered. If you attend a different section without specific direction from your lab instructor, you will be considered absent.
Laboratory Components and Grading
Participation and Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10%
Prelabs and Pop Quizzes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10%
Laboratory Notebook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20%
Mendelian Genetics Lab Report. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30%
Molecular Genetics Lab Report
Introduction and Materials & Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10%
Final Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20%
Total . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .100%
Participation and Technique (10%) – Each student begins the semester with 100 points. Points may be deducted for failure to participate, failure to follow procedures, improper disposal of materials, failure to clean up your lab space or as determined by your TA.
Pop Quizzes and Prelabs (10%) – Encourages earnest preparation for the laboratory exercise. Questions will be drawn from material presented in the lab manual. Prelabs are due at the beginning of class time. Late prelabs will result in a 10% reduction of the prelab grade.
Laboratory Notebook (20%) – A concise record of experimental data, results, and conclusions. The notebook will be evaluated twice during the semester and at the discretion of the TA. Completeness, clarity and organization, and data analyses will be checked. Late notebooks will result in a 10% reduction of the notebook grade.
You must keep a laboratory notebook containing all of the raw data you collect, along with the calculations and analyses for each exercise. A bound quad-ruled composition book must be used. Organization of the notebook is crucial. Plan ahead and decide a suitable format for your data and results before each class. Before beginning data collection, divide your notebook into sections, one section for each exercise (use section tabs). Data from different experiments should be recorded in separate notebook sections. Data can often be recorded in a tabular format to simplify for later analysis. Do not jot down data on scraps of paper and transfer it to your notebook later. All data entries should be dated and recorded in the notebook as they are collected in class.
Each section of the notebook should include all the information needed to prepare a formal laboratory report. The information should be in outline form or concise sentences, and must be arranged using the following headings: introduction, materials and methods, and results.
Introduction: the title of the exercise, a statement of purpose, and a brief explanation of the major biological and genetic principle(s) involved.
Materials and Methods: For the materials and methods used, one can often refer to the lab manual (e.g. …as described in …). However, a brief description of the methods used should always be included. It is not necessary to repeat the mechanistic details of procedures described in the lab manual nor the list of glassware or plastic ware used. Any deviations from the published protocol, including any procedural errors, should be noted.
Results: Present your data clearly and concisely in text form. This will require some forethought to ensure a meaningful and orderly presentation. Data should be presented in organized tables where appropriate. Formulas used for calculations should also be included.
Laboratory Reports (60 points) - Formal presentation of experimental data, results, and conclusions. Grading will be based on completeness, clarity and organization, data analysis and interpretation of results (see details below). Lab reports are due at the beginning of class time. A 10% reduction in the report grade will be assessed for reports handed in after class has begun, with an additional 10% reduction in the report grade for every day late up to 3 days. After three days, a grade of zero will be assigned to the report. Late reports should be turned in at the Biology Office (SCA 112) for a day and time stamp.
Plagiarism will not be tolerated and will result in a grade of zero for the report. Due to physical limitations, some experiments will be completed by students working in small groups and this means that some experimental data will be shared. This does not mean that partners may submit identical reports. Although general discussion of the exercises among students is acceptable and encouraged, the writing of reports must be done individually.
Please spell-check and proof- read your paper. Use headings for each section. Formal writing should not mirror casual conversation. Instead, it should be concise, grammatically correct, and written in third person. Use the past tense to describe experiments that were done.
Lab reports must be typed double-spaced in the following format:
Title Page (5%) – The title of the experiment, your name, your lab partner(s) name(s), the date, and the course title with section number should be included.
Introduction (25%) – Clearly state the purpose of the exercise, identify the subject, and explain the major biological or genetic principles being examined. State the hypothesis or define the problem with which the paper deals. Justify the experiments and relate them to pertinent observations or information that is known.
Materials and Methods (15%) – This section should be written in paragraph form using concise sentences. Do not include unnecessary detail, but be thorough enough so that someone else could understand and repeat the procedure. Be certain to include modifications or deviations from the protocol presented in the laboratory manual.
Results (15%) – Results should be presented clearly and concisely in complete paragraphs. In the text, call attention to the important parts of the data. Avoid discussing the meaning or interpreting your results in this section. Data should be presented in properly labeled and organized tables and figures where appropriate. These should be labeled, titles and have legends containing sufficient information so that they can stand alone. These should be included on separate pages at the end of the report. Be sure to number tables and figures consecutively, and refer to them in the text of this section and the Discussion section.
Discussion (25%) – This section is used to explain the meaning or significance of your results – interpret the data. Did your results support or negate your hypotheses or predictions? Why or why not? Any speculation should be based on sound logic and your understanding of the techniques or genetic principles involved. Unexpected results usually arise from explainable causes and should be deciphered as best as possible.
References (5%) – An alphabetical list of all reference material that has been cited in the paper. Use the following style:
Alexander, J. M. 1968. Occurrence of starches in bryophytes. Bot. Gaz. 127: 176-199.
Alexander, J. M., B. J. Martin, and T. R. Smith. 1969. Occurrence of starches in ferns. Am.
J. Bot. 47: 9-18.
Alexander, J. M. 1970. The Occurrence of Starches in Plants. Martin Press, New York. 486 pp.
Alexander, J. M. 1972. The occurrence of starches in higher plants. In: R. R. Jordon and
M. M. Taylor (eds.), Carbohydrate Reserves. Martin Press, New York. 276 pp.
In the body of the paper, information from other sources are cited in this manner:
Alexander (1968) stated that…
…was found by Alexander (1968)
…known that carbohydrates are used for reserves (Alexander 1968)
…found starch in all ferns (Alexander 1968, 1969; Alexander et al. 1969)
One author: Alexander
Two authors: Alexander and Smith
Three or more authors: Alexander et al.
Format (10 %: Lab reports must be typed and double-spaced. Use 12 point font. Maximum length excluding tables, figures and references is ten pages.
Mendelian Genetics Report – This report should cover the Mendelian Genetics module including dihybrid crosses and genetic mapping. It should not exceed 10 typwritten double-spaced pages excluding figures and tables..
Molecular Genetics Report – This report covers all of the Molecular Genetics Module exercises and will be separated into two parts. The first part will cover only the Introduction and Materials and Methods. It should follow the report guidelines for these sections outlined above. A reference page should be included, and references should be cited properly in the text.
The second part will cover the full report. It will be graded according to the guidelines . The maximum number of text pages excluding tables or figures should be 10 pages for the full report. Corrections to the Introduction and Materials and Methods from the first part will be expected.
Pipetting techniques
Pipetting techniques are very important. Mistakes can result in failure of experiments and waste of reagents. The Rainin pipetman pipettor is the industry standard in Biotechnology. Each pipettor is a precision instrument that costs nearly $300. Do not drop or mishandle the pipettor.
Ranges of Accuracy:
There are three sizes, each adjustable within a range:
P-1000 Useful for 200 to 1000 microliters
P-200 Useful for 20 - 200 microliters
P-20 Useful for 1 - 20 microliters
Never use a pipettor in a range it was not designed to measure accurately. For example, if you need to measure 18 ul, the temptation is to use the P-200 because it is fastest to adjust, but a more accurate measurement could be made with the P-20. In the extreme, one could conceivably try to use the P-1000 to pipette 1 microliter, but obviously, this would be very inaccurate. The P-1000 uses large blue disposable tips, the P-200 and P-20 use smaller yellow tips.
Volume adjustments:
There are three digits for each pipettor, so set them accordingly:
Top digit Middle digit Bottom digit
P-1000 thousands hundreds tens
P-200 hundreds tens ones
P-20 tens ones tenths
Drawing a sample into the pipettor:
Place a tip on the pipettor by gently but firmly pushing the pipettor barrel into a tip in the rack. After you have set the volume you need, Push on the white plunger with your thumb and slowly push it down until you feel resistance (the first “stop”). Pushing further than the initial resistance activates the blow out feature and would result in too much liquid being drawn into the pipettor. place the tip just below (2-3 mm) the surface of the liquid you are using. Do not jam the pipette tip all the way to the bottom of the container! Slowly draw the sample into the tip by gently relaxing downward pressure on the plunger. Watch carefully to see that the sample is slowly drawn into the tip. Do not release the plunger all at once or the measurement will be inaccurate and likely contaminate your sample and damage the pipettor.
Move the tip to the tube to which you want to transfer the sample. Place the pipette tip against the inside wall of the tube near the bottom but just above any other liquid already there. If the tube is empty, keep the tip a few mm above the bottom. Slowly push the plunger to the first “stop” and check to see that the drop slowly emerges from the tip and adheres to the tube’s inner wall. Then firmly push the plunger to the second stop to blow out any liquid remaining in the tip.
Expert biotechnicians learn to watch how much liquid is in the tip and mentally estimate its volume by this observation. They would quickly notice if they had a major error. For example if they intended to pipette 2 microliters but accidently drew 10 microliters into the tip, they would notice the problem in time to correct the error. Experts always watch the drawing of liquid into the tip and watch that it is properly expelled into the tube so they notice problems in time to correct errors.
Inexperienced students often withdraw too much into the tip because they pushed the plunger past the first stop prior to drawing a sample into the pipette. Another common mistake is to use the wrong pipettor for the range to be measured, or set the pipette volume incorrectly to begin with. Another common problem is not pipetting carefully and deliberately. Always watch the liquid being drawn up and watch it carefully when it is expelled. Mentally estimate if the volume in the pipette is in the right ballpark of the volume you set.
Ejecting the tip:
To avoid cross contaminating samples and reagents, use a fresh tip for each pipetting action. There is a small plunger that moves the chrome plated tip ejector which extends around the barrel of the pipettor. Pushing firmly on this plunger causes the pipette tip to fly off from the pipettor. Position it over the sharps box before you eject the tip.
Pipette techniques data analysis
Each student will receive two dry microfuge tubes. These should be labeled A and B and have initials of the student. Weigh them to the nearest one/hundredth of a gram (0.01). Record this number in your lab book.
Mock reagents will be supplied in other microfuge tubes:
Enzyme
10X buffer
Water
DNA
Make up the following reaction in Tube A:
100 ul 10X buffer
893 ul Water
5 ul enzyme
2 ul DNA
Close the cap and weigh to the nearest 0.01 gram.
Make up the following reaction in Tube B:
10 ul 10X buffer
87 ul Water
1 ul enzyme
2 ul DNA
Close the cap and weigh to the nearest 0.01 gram.
Analysis:
Mass of tube A with reagents: _________
Mass of tube A without reagents -_________
Difference tube A _________
Mass of tube B with reagents: _________
Mass of tube B without reagents -_________
Difference tube B _________
Assume that all the mock reagents have a density equal to that of water. Calculate the volume represented by the weight of the reagents in the tube (in other words every 1/100 of a gram equals 1 microliter). This number will be the observed volume of reagents.
Observed volume tube A ________
Observed volume tube B ________
Calculate the total volume you were told to pipette into each tube. This number will be your expected volume of reagents. Calculate your percent error as described below:
Expected volume tube A ________
Expected volume tube B ________
(Observed volume - expected volume)/expected volume X 100 = percent error
If your error was greater than 5%, obtain new dry tubes, reread the directions above very carefully, ask you TA for advice, and repeat the exercise.
Laboratory Safety Rules
Safety glasses must be worn when any chemical, in any amount, is used - including preservatives and stains or where there is the possibility of an object impacting the eye.
All pagers and cell phones must be turned off before class begins.
Shoes must be worn at all times. No flip-flops / sandals.
Lab coats and long pants are recommended to protect you and your clothing.
Long hair should be tied back.
No food, drinks or smoking in lab.
If you have an accident at your station, notify your instructor immediately.
If you are bleeding, notify your instructor immediately and remain at your station.
Do not leave working equipment unattended.
Dispose of all broken glass in the “broken glass” box.
Dispose of all biohazardous material in the biohazard bag.
Non-absorbent material (i.e. glass) that has been contaminated must be placed in the “sharps” box.
Used pipettes will be collected in a special box during those labs in which they are used.
Wash your hands immediately after the laboratory session.
Wipe your workbench with disinfectant after the laboratory session.
Book bags and purses must be stored in the place designated by your instructor
PCB 3063L General Genetics Laboratory Lab Schedule
Week of… Mendelian Genetics Module Molecular Genetics Module
Aug28 Introduction to Course, Statistics
Sept 4 Introduction to Drosophila
Sept 11 Basic Mendelian Inheritance in Drosophila
Sept 18 Recombination and Mapping in Drosophila
Sept 25 Data Analysis Pipetting techniques
Oct 2 Data Analysis
Oct 9 Lab Report 1 and notebook due. Genomic DNA extraction
PCR amplification
Oct 16 Purification and quantification Cycle sequencing
Oct 23 Cycle sequencing clean-up
submit for sequencing Set-up PCR for cloning
Oct 30
Clone with Topo kit: Ligation and
transformation
Nov 6 Pick colonies
Blast search in computer lab
Nov 13 Mini Preps and digestion
Nov 20 Restriction Mapping
Nov 27 Thanksgiving Holiday
Dec 4 Catch up Lab
Report 2 and notebook due.