METHODS IN MOLECULAR BIOCHEMISTRY LABORATORY
BIOCHEMISTRY 526: Fall 2010
OBJECTIVES
This course is designed to provide a foundation experience for conducting experiments in a
biochemistry laboratory. Experiments/techniques include spectrophotometric quantitation of
DNA and proteins, yeast genomic DNA and complementation analysis, transformation of
bacteria and yeast, plant gene expression analysis by RT-PCR, molecular cloning, protein
expression and purification, and kinetic analysis of enzymes and inhibitors. In short, you will
leave this lab with many experimental techniques used in academic and industrial laboratories
that will benefit you throughout your scientific career (and that you can list on your resume!).
The main objectives of this course are to:
1. Learn how to collect, record and analyze experimental data.
2. Become familiar with some of the equipment, methodologies, and instruments used in
biochemistry laboratories.
3. Present results clearly in written and verbal formats while adhering to the standards and style
used by life scientists.
4. Perform experiments in an environment requiring teamwork.
EXPERIMENTS
Experiments to be performed in Biochemistry 526 are contained in this manual. The appropriate
background information should be read prior to the laboratory period in which the experiment
will be conducted. The experimental procedure sections should be studied in detail, and you
must understand the reasons why the major steps are being performed before coming to work in
the lab. You may be given a prelab quiz at the start of lecture that will be graded. All
experiments will be conducted in pairs unless otherwise noted. You and your lab partner
perform the experiments jointly, but each maintains her/his own lab notebook and independently
writes lab reports.
ATTENDANCE
The class meets on Tuesdays and Thursday afternoons as indicated in the syllabus. Attendance
is mandatory. Lectures will be given in ISB 329, and labwork will be conducted in ISB 268.
Class begins promptly! Please be on time; no makeup is available! Also, you will receive
points for attendance that CANNOT be made up. There will be 26 PLQ/assignment and 26
attendance grades (0 or 5 pts); only the lowest will be dropped and failure to attend for
ANY reason will result in a zero for both on that lab day. One unexcused absence is grounds
for being dropped from the course. Serious, unavoidable conflicts should be brought to the
attention of the instructor prior to missing the laboratory period. Extended absences can result in
withdrawal.
Any student requiring special accommodations MUST contact the instructor in the first two
weeks of lab, and MUST present an official, signed letter detailing the accommodations to be
required.
ASSIGNMENTS
1. At the start of lecture you may be given a prelab quiz (PLQ) that covers the experiments to
be conducted during that day’s laboratory period. Completion of pre-lab assignments will also
be checked and graded throughout the semester. All prelab quizzes will be administered at the
start of lecture, and you will not receive credit for the PLQ if you arrive late to lecture.
2. A major goal of this course is to teach the students how to represent laboratory data in a
format that is used in professional life sciences journal articles. You will be expected to report
your data in proper format for lab reports. Use examples provided to help you make graphs,
write figure legends, and format your tables. In presenting quantitative results, consider
carefully how many significant figures are justified. All laboratory reports will be wordprocessed, and are due as indicated in the manual or as announced in class. These assignments
constitute part of the Junior Year Writing Requirement. Failure to submit any assignment will
result in no credit for that assignment. The official class policy on late assignments is below.
LATE POLICY FOR BIOCHEM 526 ASSIGNMENTS:
PLQs/pre-lab assignments: Administered/due at the start of lecture or ZERO POINTS if late.
Lab Reports: Due at the start of lecture or…
if turned in during lecture, end of lecture, or in/after lab that day…10% of full point value off
if turned the following day
… 10% of full point value off
two days late
… 20% of full point value off
three days late
… 30% of full point value off
four days late
… 40% of full point value off
five days late
… 50% of full point value off
more than five days late
…zero for that assignment
Note that skipping a lecture or lab to complete an assignment will result in no credit for that
day’s work, and your assignment will still be late. Additionally, failing to show up for a lecture
or lab (or late arrival) will result in the student not attending lab, with no make-up available.
Failure to contact a lab partner, record data, busy schedule, other classes, etc. are not acceptable
reasons to be late on assignments! Extensions are ONLY for truly extenuating circumstances
with appropriate official documentation, regardless of whether they are requested in
advance or not.
RECORDING DATA
All essential data are to be recorded in a suitable research notebook available at bookstores.
These data include but are not limited to protocols, identification of samples, and all primary
observations. Do not write on scraps of paper. The recording and organization of a permanent
record of laboratory observations is as important a technique to master as any of the
experimental methods taught in this course. The research notebook is a day-by-day record of the
progress of your experimental work and should reflect the sincerity and honesty of the
experimenter. Write everything down clearly. Remember, sometime later you (or someone
else) might have to go back and rely on your notes to figure out what you did!
POLICY ON PLAGIARISM
Plagiarism covers a range of misrepresentations, many of which are easily avoidable by proper
use of citations and good writing skills. The academic honesty policy at the University of
Massachusetts Amherst defines plagiarism as: "...knowingly representing the words or ideas
of another as one's own work in any academic exercise."
Please refer to the following website for these policies:
www.umass.edu/dean_students/code_conduct/acad_honest.htm
For this class, specific examples of plagiarism include:
1) failure to provide a citation (reference) for data/information obtained from another source
2) directly quoting from a cited source without using quotation marks
3) closely paraphrasing a cited source
4) using the same or very similar wording as another student in the class
5) using data tables, figures, legends, or text generated by or with another student in the lab
6) copying another student’s PLQ, Exam, or Lab Report
7) distributing your work (e.g., lab report) by any means, including electronically
8) copying or closely paraphrasing a lab report generated during a previous semester
Plagiarism is not acceptable and will result in NO credit for the assignment, possible
withdrawl from the class, and notification of the Academic Honesty Board. NO
EXCEPTIONS!
LABORATORY CONDUCT
1. Much of the equipment used in this course is expensive and none of it is insured. If you
have trouble with the operation of an instrument, do not hesitate to ask the instructor for help.
2. Never bring food or beverage into the laboratory. Do not dispose of food wrappers or
beverage containers in the lab wastebaskets, even if the contents were not consumed in the lab.
3. Work safely in the lab. You are required to wear eye protection in the laboratory at all
times. Wear proper eye protection before turning on any UV light. Place backpacks, coats,
jackets and sweaters in the available shelf space to avoid fire and chemical spill hazards. Note
the locations of First Aid kits, fire extinguishers, and drenching showers.
4. Always think ahead. Prepare for the next step in the procedure while waiting.
5. Make sure you understand what you are doing. If you do not, feel free to ask.
6. Keep your bench area, balances and instruments clean and clutter-free. Wash your hands
before leaving the lab.
7. Cell phones and music headsets cannot be used in the laboratory.
Additional note: when an experimental procedure calls for addition of water, you should
only be using sterile distilled water, provided in bottles.
SUMMARY
Biochem 526 IS likely your most demanding class, and is designed to be your top academic
priority. This class should also be the best and most enjoyable biochemistry experience at
UMass, and it is my job to prepare you for life as a biochemist in academia or industry.
The skills you are learning are universal, and will be required in the real world! One vital
skill (see course objectives above) you will learn is teamwork and partnership. Be sure you
and your lab partner are aware of what each other is doing, as you are sharing all data and
observations, upon which your results, interpretations, and grades are based!
BIOCHEMISTRY 526 GRADING PLAN
Instructor reserves the right to edit grading scheme
PLQs/assignments
LR1
LR2
LR3
LR4
LR5
10 pts each
600 points total for semester
100 points total
100 points
100 points
100 points
100 points
100 points
Biochemistry 526 FALL 2010 Tentative!
Tuesdays
September 7
Micropipetting and liquid handling; assessing the
concentration of biological molecules
September 14
Inhibitor screening using high-throughput
instrumentation (“the robot”)
September 21
Experiment 2: Isolate yeast genomic DNA,
determine concentration, restriction digestion, PCR
September 28
miniprep plasmids from bacteria; determine
concentration; transform yeast
October 5
plate yeast on Fe medium; determine transformation
efficiency; mate his- strains
October 12
NO LABORATORY TODAYMONDAY CLASS SCHEDULE FOLLOWED
October 19
Stress plants, isolate/quantify RNA; perform RT-PCR
Run gels to validate primers
LAB REPORT for Yeast Molecular Biology DUE
October 26
Student presentations of RT-PCR data
type and duration of stress, timepoints, analysis of
results, questions
November 2
Ligate DNA and transform bacteria
Wednesday, November 4: count colonies, patch cells
and begin PCR screen; lab open 10:30-12:30
LAB REPORT for Plant Stress DUE
November 9
perform minipreps from selected colonies, obtain
A260/280, set up restriction digests, run gels
November 16
Experiment 5: expression analysis: IPTG induction,
B-gal assays
November 23
Mandatory data analysis discussion period
November 30
Run SDS-PAGE gels of purification samples; transfer
proteins to membrane
December 7
Investigating enzyme structure/function relationships:
a lecture by Prof. Jeanne Hardy; evaluations and
semester wrap-up
Thursdays
September 9
Experiment 1: Dependence of Vo of AChE upon time
and [E]; determination of Vo vs. [S]
September 16
Assessing progressivity of inhibitors and determining
mechanism of inhibition
September 23
Agarose gel electrophoresis of digests and PCR
LAB REPORT for Enzyme Kinetics DUE
September 30
plate yeast transformations
October 7
plate diploids on –His medium
Experiment 3: The Arabidopsis Information Resource
(TAIR); design primers for RT-PCR; submission of
detailed plant stress experimental design
October 14
Obtain plant genomic DNA and test primers by PCR
observe functional complementation on Fe medium
plates; observe genetic complementation in diploids
October 21
Agarose gel electrophoresis and analysis of RT-PCR
October 28
Experiment 4: Digest plasmids, run agarose gel and
purify fragments, phosphatase-treat vector
November 4
Run PCR screen on gels and set up cultures
November 10 (class meets on Wednesday!)
Design of protein expression and purification
experiment
November 18
Ni column chromatography; B-gal activity assays and
BCA with fractions
LAB REPORT for Molecular Cloning DUE
November 25
NO LAB- TURKEY AND 3 NFL GAMES!!!
December 2
Immunoblotting; analyze SDS-PAGE gels and blots
December 9
LAB REPORT for Protein Expression/Purification
DUE