INTRODUCTION TO BCH452
Biochemistry 452, 2010
Dennis Brown, PhD
E-mail:
dennis_brown@ncsu.edu
Trino Ascencio-Ibáñez, PhD
E-mail:
trino_ascencio@ncsu.edu
TA for BCH 452
Andy Caldwel: accaldwe@ncsu.edu
E-mail
Major mechanism of communication
Notify you of changes in schedule and project
Send updates on experiment results
Answer your questions
Keep your mail box clean
Lecture notes on web are too large to send
Some Mails may still push you over quota
Lab Text
BCH 452 “ Introductory Biochemistry”
Author: Charles Hardin & James Knopp.
NCSU
Available at Sir Speedy
2526 Hillsborough ST
$25.50 (approximate)
Schedule
Lab opens at 1:15
Turn in Pre lab questions & lab reports
Collect any handouts
1.30 lecture
2:00 lab exercise
NO MAKE UP LABS
Please be aware of this
BCH 452
Will introduce fundamental techniques in
biochemical analysis
Will teach the principles upon which the
technology depends
Will make you familiar with theory and
practice of the technology
More Importantly BCH 452
Will stress proper laboratory technique
Reproducibility
Understand and eliminate errors
Will stress proper record keeping and data
reporting
Necessary for archival considerations
For intellectual property
For trouble-shooting
Concept of 452
Teach techniques in common use in
biochemistry laboratory science
Try to present situations that exist in a real
laboratory situation
Move from “exercises” with predictable outcomes.
To “experiments” with doubtful outcomes
Important to understand and explain
anomalous results
My Job…
To be certain that you understand the
principles that the experiments will examine
To be certain that you understand how the
apparatus employed in the experiment
functions
To be certain that you understand the reason
for each step in the experimental protocol
Lab Safety (Front of Lab Manual)
Hazardous reagents
Volatiles
Corrosives
Mutagens
Safety goggles
Eyewash, safety showers, fire extinguishers,
sharps containers
Exits
NO OPEN TOE SHOES
Model Molecules
Three major classes of biomolecules
Carbohydrates
Proteins
Nucleic acids
Model Techniques
Spectrophotometry
Chromatography
Electrophoresis
Biochemical fractionation
PCR and qPCR
ELISA
Your Responsibility
Read the experiment before you come to lab
Read pertaining sections of appendix
Read “Tips”
Turn in the pre lab exercise
Have all required materials
Some of the protocols can be confusing.
Make a flow sheet that outlines the steps.
Grading
Lab reports: 130 total points
Pre Lab Questions: 36 points
Exams (2): 25 points each
Total 216 points
A+216 - 205 C+ 150 - 140
A 204 - 193 C 139 - 130
A- 193 - 184 C- 129 - 119
B+183 - 173 D 118 - 108
B 172 - 162 F 107 - 0
B- 161 - 151
Reporting
Prelab questions
Due at beginning of designated lab
Ensures that you have read and understand the exercise
Completed report
Due at beginning of next exercise
Laboratory Notebook (Ideal)
No loose pages
Generates copies
All entries dated
Each page dated
Pages signed by a witness attesting to date
(extreme)
Retention time (7 years)
Will provide extra credit if in good shape
Lab Reports should contain
Statement of purpose
Why you are doing the experiment
What you wish to determine
Brief account of theory and design of experiment
Summary or flow chart
The raw data
All calculations
Results
Conclusions and interpretation
Lab reports are personal
You should write your reports as a
personal effort, even if all the data and
the experiments are performed as a
group.
A form for lab write up in lab
manual
Title all sections in the manuscript
Abstract, Discussion, Graph titles, etc.
Abstract should contain a brief summary of the
experiment
“I investigated the effect of temperature on the
activity of the enzyme Stickase by monitoring its
ability to convert stick to broken stick. This was
done by determining the accumulation of broken
stick by spectrophotometry (A260) at temperatures
between 10 to 60 degrees C. I found that Stickase
converted stick to broken stick most efficiently at a
temperature of 37oC”
Lab Report (read information in manual)
Record all your experimental data
including changes in protocol
Include your calculations and raw data
as appendix attached to your report.
Label co ordinates. Be careful with units,
spelling and grammar
Explain sources of error
Things that you should know
Units of Biochemistry
Decade system (Metric-decimal system)
Moles
Meters
Liters
Grams
Centigrades
Elementary calculations
Gram Molecular Weight
Dilutions (very important in lab
environment)
Rate equations
“What is the molar concentration of a
solution that is 5 millimolar NaCl”
Significant Figures
Determines the value of a data set
Includes estimations
3.2 is not the same as 3.20 or 3.200
Presentation of Data
Data tables
Graphs
A Table or Graph should:
Be easy to understand
Project a message
Does not require extensive reference to
text
Experiment
A solution of compound X is reacted with
reagent Y which causes the production of a
light absorbing product. The amount of light
absorbing product indicates how much
compound X is present. The sample
containing X is divided into three aliquots and
the experiment is repeated three times at
three wavelengths.
Lousy Table
Results of the three experiments
Absorbance values Absorbance values Absorbance values
for reaction #1 at for reaction #2 at for reaction #3 at
360, 420 and 540 360, 420 and 540 360, 420 and 540
nm nm nm
0.875 (360) 0.885 (360) 0.823 (360)
0.253 (420) 0.250 (420) 0.244 (420)
0.164 (540) 0.165 (540) 0.157 (540)
Good Table
Absorbance values for the “experiments” at 360,420 and
540 nm
Trial # A360 A420 A540
1 0.876 0.253 0.164
2 0.885 0.250 0.163
3 0.825 0.244 0.157
Bar Graph A: Compares Values at each
wavelength, Shows Reproducibility
0.9
0.8
0.7
0.6
0.5
Absorbance Trial 1
0.4
0.3 Trial 2
0.2 Trial 3
0.1
0
A360 A420 A540
Wavelength
Bar Graph B: Compares absorbance at different
wavelength, shows sensitivity
0.9
0.8
0.7
0.6
0.5 A360
Absorbance A420
0.4
0.3 A540
0.2
0.1
0
Trial 1 Trial 2 Trial 3
Next Exercise (January 25th, 26th,
27th)
Carbohydrates
Section A1 in lab manual
Read carefully prior to coming to lab.
Do the pre lab questions and turn them in
at your arrival.
If you don’t know how to use a
micropipette:
Stay today to get trained, this task is
extremely important for the good
practice of Biochemistry and therefore,
your grade in BCH452.