Bio366L Manual v56 S2022.docxBIOLOGY 366L
Table of Contents
Introduction to the Laboratory …………………………………………………………………………………………….. 2
Exercise 1
Basic Lab Skills ……………………………………………………………………………………………….. 11
Exercise 2
Enzymatic Reactions ………………………………………………………………………………………. 27
Exercise 3
Basic Cloning Methods ……………………………………………………………………………………. 35
Exercise 4
Clone Verification…………………………………………………………………………………………… 49
Exercise 5
BioInformatics ……………………………………………………………………………………………….. 59
Exercise 6
Bacteriophage Discovery………………………………………………………………………………… 62
Appendix 1
Excel 2011 for the MAC …………………………………………………………………………………. 91
Appendix 2
ImageJ Application………………………………………………………………………………………… 95
Appendix 3
Photoshop Application ………………………………………………………………………………….. 99
Appendix 4 PCR Purification …………………………………………………………………………………………… 106
Appendix 5
DNA Extraction from Agarose Gels ………………………………………………………………… 108
Appendix 6
Laboratory Safety ……………………………………………………………………………………….. 109
Appendix 7
Lab Report Guidelines………………………………………………………………………………….. 114
Appendix 8
Reagents and Solutions ……………………………………………………………………………….. 117
Appendix 9
Supply Locker……………………………………………………………………………………………… 128
Appendix 10
Standard Operating Procedure (SOP) for Pipetman………………………………………… 129
Introduction to the Laboratory
Page 2
Introduction to the Laboratory
Due to the nature of the COVID-19 pandemic, the University seeks to provide flexibility and
safety to the students, staff, and faculty. Some of the information in the lab manual may not
apply for the 2022S semester in a literal sense. Please refer to the course syllabus for the most
recent, up-to-date information. In the case that the syllabus and the manual differ in wording of
policies and rules, the syllabus will be used as the standard.
Laboratory organization
A group (up to four) of students will be issued keys to a locker containing glassware and other
supplies. A Locker Supply List with quantity and cost of each item will be provided to each group.
Please check to see whether all the supplies listed are in fact present in the locker (See Appendix
9). If there is any discrepancy, bring it to the attention of your lab instructor. If everything is in
order, please put the locker number on the Locker Supply List with all members of the group sign
the paper and return it to your lab instructor. At the end of the semester the locker contents will
be checked again. Also sign out for the keys on the key card and return the key card to your
instructor. Keys need to be returned at the end of the semester. Lost keys will result in a charge of
$10 per key. Please keep the spectrophotometer tubes and cuvettes in the wooden racks only;
metal racks scratch them. Please don’t store liquid in open containers in the locker. The liquid
might leak/spill, causing the wood of the locker to swell, then the locker cannot be opened.
Additionally, each group of students will be issued keys to another locker for storing their lab coats.
Sign out for the keys on a key card and return it to your instructor. Usually, a group of students will
be issued four keys; each student (in the group of four) should hold on to one key per individual.
Only write down the key ID on the key card if the locker key is in your possession. The key cards
will be used to account for keys returned at the end of the semester. If you write down a key ID on
the key card that is not in your possession, you will be responsible for it at the end of the semester.
Many of the solutions you will need are provided in bottles in the middle of the benches. The two
groups of students sharing the same bench will also share these bottles of reagents. Determine
how much you will require for a particular exercise, bring your own labeled beaker or flask aliquot
out the approximate amount needed. It is not good practice to return left over portions of
solutions to the original bottle. When reagents are available in small quantities (< 1.0 mL) you
should use the amount that you need directly from the bottle or tube. All containers that are not
empty should be labeled. Labels on chemicals and solutions that will be saved should include all
known components in the container, the concentration of each component, possible hazard
associated with the chemicals, the student group number, the lab section number.
Specialty glassware and material will sometimes be needed for certain exercises. Use this during
the lab period, clean it, and return it so other classes can use the material as well. Please DO
NOT put them into your locker. There will be a negative impact to your subjective grade if you
sequester the shared equipment/glassware in your locker. In addition, please do not write on
these shared equipment and glassware. If necessary, write on a piece of labeling tap and attach
the tap to the equipment. If you do not know how to use certain items of equipment such as pH
Introduction to the Laboratory
Page 3
meters, centrifuges, spectrophotometers or microscopes, please be sure to get instructions on
their use either from the lab syllabus or from your laboratory instructor. Unless instructed
otherwise, all equipment such as spectrophotometers and water baths should be turned off at the
end of the laboratory period.
Broken glassware and other sharp objects should be placed in the sharp objects container provided
in the laboratory. Hazardous solutions should be disposed of in the appropriate waste bottles
found on the laboratory benches. All gloves should be disposed of in the plastic biohazard
container. This container may be opened by stepping on the foot pedal and should never be used
to dispose of items unless there is a biohazard bag inside. Never ingest any solutions or chemicals
in the lab and wear gloves if you must contact solutions or chemicals in the lab.
Please be aware of the following general guidelines for measuring volumes of solutions. When the
volume to be measured is greater than 20 mL, use a graduated cylinder. If the volume is between
5.0 mL and 20 mL, use 5, or 10 mL serological pipettes; whichever is closest to the volume you
need. Serological pipettes are provided in the lab and when you are through using them, please
put them in the appropriate “used pipette waste container.” Learn to use the Fisher Pipette
Controller to fill the serological pipettes. Never mouth pipet! For volumes of 0.002 mL to 1.0 mL
use the Gilson Pipetman (or equivalent) with the plastic tips. Your lab instructor will demonstrate
the proper use of the Fisher Pipette Controller and the Gilson Pipetman.
Reverse osmosis (RO) water is available from the faucets at the classroom sinks. At the classroom
sinks, there are two faucets for each sink; one for regular tap water and the other RO water. Please
know the difference between the two. Regular tap water faucets have 2 handles (one for hot
water; one for cold water). RO water faucets only have one handle (cold). When water is called for
in the lab manual, use RO water unless instructed differently. Also, when washing glassware, do a
final rinsing of equipment/glassware in RO water to prevent hard water residue build-up.
The laboratory exercises
Each student will conduct six exercises during the semester. You will usually work in groups of (up
to) four on these exercises. Exercises 1 and 2 are designed to help you brush up on your laboratory
skills, especially your sterile techniques and your ability to pipet accurately. Exercises 3, 4, and 5
combined to make one large cloning experiment. Exercise 6 is an exploratory experiment where, if
successful, you’ll be able to discover a never before seen/identified bacteriophage.
In the classroom, there is a large whiteboard. Instructions will be written on the whiteboard
specific to each experiment to be performed for that day. Please read and follow these
instructions to make sure successful completion of experiments for the day.
While doing the exercises, please do not treat the manual like a cook book where you follow the
steps in sequential order. Sometimes, important information is giving at the end of a section;
please read the entire protocol first before staring the experiment.
Page 4
Introduction to the Laboratory
The laboratory notebook, written reports, and quizzes
Hopefully, you have already learned to keep accurate and neat records in a permanent laboratory
notebook. Also keep all calculations, graphs, and conclusions in this notebook. Exercises 1 and 2
are designed to get you into the habit of keeping a good lab notebook. The notebook of choice is
the one listed in the supply list at the bookstore consisting of alternating white and blue pages of
1/4" graph paper (National Brand-Laboratory Research Notebook #43-649).
The notebook will include well labeled original data usually in the form of numerical tables or
sometimes in the form of statements, derived results based on the original data such as
calculations or rate determinations for example, summary graphs and summary tables. Use a piece
of carbon paper to generate a copy of your original data and turn this copy in at the end of each lab
period. Your instructor may also wish you to turn in copies of your derived results, summary
graphs, and summary tables from your notebook with your written report. Do not erase mistakessimply cross them out with a single line and continue. If you work at a company and patent your
findings, erasures can invalidate the patent application.
A reminder or two about graphing – Graphs are for establishing or illustrating a relationship
between two variables. One variable is usually the independent variable and is placed on the X-axis
(abscissa). The other variable is the dependent variable and is placed on the Y-axis (ordinate). Each
axis is usually a linear scale and should be labeled with units and what it represents. The X, Y
intercept need not be zero for each axis. Make the X, Y intercept zero only if it is critical to
demonstrate a value of Y when X is zero. See Exercises 1 and 2 for examples of graphing where
absorbance (or transmittance) is dependent upon wavelength, absorbance (or transmittance) is
dependent upon solute concentration, absorbance is dependent upon time, etc.
You will be asked to submit one written reports. You are asked to submit individual reports (i.e.
each person will have a different report). Each report will include an Introduction that describes
the basis of the study and defines what is to be reported. The second part will be Materials and
Methods that should include methods that are not already presented in the lab manual (please do
not copy the methods given in the lab manual into your report; that is wasted effort). The third
and most extensive part of the report is the Results section that includes well labeled original
results usually in the form of numerical tables or sometimes in the form of descriptive statements,
derived results based on the original data such as calculations or rate determinations for example,
summary graphs and summary tables. For each summary graph or summary table a one paragraph
discussion should be given. This discussion should state the meaning of the summary graph or
table, compare the result you obtained to some expected result and evaluate your result. The
fourth section is the Conclusion, where you take all the data and results from your experiment to
formulate an answer to your original thesis. The final section of the paper is Literature Cited
section that gives complete references to those papers or books cited usually in the introduction
and conclusions. See Appendix 7 of this lab manual for further guidelines on writing your reports.
Reports will be checked for plagiarism via the “Turnitin” electronic service. The Turnitin electronic
service will compare your report to all the previous submitted reports that are in their database.
The Turnitin service will highlight the parts of your report that are exactly the same or similar to
Introduction to the Laboratory
Page 5
other reports and provide a similarity score. A similarity score of more than 50 will trigger an
automatic investigation where the report will be turn over to the Center for Student Rights and
Responsibilities (http://go.sdsu.edu/student_affairs/srr/Default.aspx) without notifying the student
first.
Grading
Course final grades will be based on the homework, quizzes, and assignments detailed in the table
below. Due dates for the various quizzes and assignments are listed in the class schedule at the
end of the syllabus. Late assignments/quizzes will not be accepted and will be giving a point value
of zero.
Lab clean-up and etiquette (adapted from the Bio203L syllabus)
Students must clean up their work areas at the end of each lab session and return equipment and
materials to storage locations. This is important for reasons of safety and to clear the space for the
next class. You have a right to a clean, uncontaminated work station, and so does the next person
after you. It’s about professionalism, responsibility, and respect for your community. If you have
any complaints about the condition of the lab, report it to your TA, or to the lab coordinator. Points
may be taken from entire classes or individual students who consistently neglect to clean their
workstations.
In the case where you need to label glassware and shared equipment, please write on a piece of
labeling tap and attach it to the glassware/shared equipment. DO NOT write on the glassware and
the equipment directly. Once you are done using the shared equipment/glassware, please wash
them thoroughly and do a final RO water rinse before returning them back to the podium for the
next class. Please do not put the shared glassware/equipment into your locker. There will be a
negative impact to your subjective grade if you sequester the shared equipment/glassware in
your locker.
There will be many instances where a single bottle or a single tube of reagent will be shared
between the entire class. The shared reagents will be left up front by the podium. Please bring
your tube and a pipette to the front podium to get the amount you need. Do not take the shared
reagent bottle back to your bench.
When you need to store tubes and petri dishes in the classroom incubator or refrigerator, please
write your section number (S#) and group number (G#) on the tube/petri dish. Items will be
thrown away it they are found without identifying numbers indication the student’s section and
group.
Lastly, when you need to exit the classroom, please take off your lab coat and gloves. Only the
individuals who are actively pipetting or physically involved with handling the reagents need to
wear gloves. Gloves are considered contaminated and you should not be typing on the lab
computer, writing in your notebook, playing with your cell phone, or touching the door knob
Introduction to the Laboratory
Page 6
with gloves on. Please be conservative with the glove usage to minimize waste, but do protect
yourself when you are actively working and handling potentially dangerous reagents/solutions.
Grade Weighting
Virtual Week 1
Virtual Week 2
Exercise 1
Virtual Week 3
Weighing Rubric
(quiz 1) Solution making & Math 15 points
(homework 1) Math & calculations 10
(quiz 2) Buffer & pH 15
(quiz 3) Pipetting 20
(quiz 4) Exercise 1 18
(homework 2) MOPS buffer 10
(quiz 5) Enzyme basics & kinetic 15
Virtual Week 4 (quiz 6) Cloning & electrophoresis 15
(quiz 7) Exercise 2 18
Exercise 2
(homework 3) AP inhibitor 10
Exercise 3 (quiz 8) Exercise 3 18
Exercise 4 (homework 4) DNA standard Curve 10
Virtual Week 5 (quiz 9) Nucleic acid & gene expression 15
Virtual Week 6 (quiz 10) Protein basics 15
Written report 25 (on Exercises 2)
Oral presentation 20 (Exercise/topic to be assigned)
Pre-labs 18
Subjective evaluation 20
Total 287
The subjective evaluation of your lab work will contribute a maximum of 20 points toward your
overall grade. The subjective grade will be assigned by your laboratory instructor and may be
based on factors such as contribution to group effort, leadership, enthusiasm, precision in
executing exercises, following lab rules and safety precautions, clean-up, attendance, and
preparation for each lab session. Each student will start his/her subjective points at 15 and go up
or down from there. In order to go up, a student must demonstrate leadership ability to help
fellow students learn or help the instructor teach.
A general guideline for the subjective portion is as follows:
20
Truly exceptional
18
Very good
Always worked hard, showed extensive preparation for
classes, group leader, active participant throughout the class.
Facilitate understanding of class materials.
Same criteria as above, but maybe a little weak in one or more
areas. Help TA with classroom clean up.
Introduction to the Laboratory
15
Good/Average
10
Below average
5
Fair
Page 7
Missing one of the criteria above. Turn in assignments on
time. Did everything a student is expected to do.
1 unexcused absents, arrive late and/or left early, missing 2 of
the above criteria. Turn in an assignment late. Not turning in
data/materials at the end of class. Leaving workspace dirty.
Breaking common equipment. Not turning in locker key.
Missing all criteria above. Turn in assignments late.
2 or more unexcused absents will result in an “F” for the semester;
3 or more excused absents will result in an “F” for the semester;
3 absences in any combination will result in an “F” for the semester.
If the written report is evaluated as unsatisfactory, it may be corrected and resubmitted within one
week. If the revised version is satisfactory, credit can be given for the report but the maximum
number of points will be 30% less than the original number of maximum possible points. Student
will have 7 days (including Sat & Sun) to submit the revised version. The written report will be
considered late if it is not turned in by the beginning of the class period on the day they are due.
Late report will be penalized three points for each day it is late, up to a maximum of 15 points. The
written report will not be accepted more than 5 days late and unaccepted report will be given a
grade of 0 points.
Student report will be checked for plagiarism using the “Turnitin” plagiarism prevention service.
Students who plagiarize material will be penalized with an F grade in the course and will be
reported to the Student Rights and Responsibility Center. See the course syllabus for more details
regarding plagiarism.
Below is the scale used for the class:
A ............. 93 – 100+ %
A90 – 92.9 %
B+
87 – 89.9 %
B ............. 83 – 86.9 %
B80 – 82.9 %
C+
77 – 79.9 %
C ............. 73 – 76.9 %
C70 – 72.9 %
D+
67 – 69.9 %
D ............. 63 – 66.9 %
D60 – 62.9 %
F ..............
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