Lab Methods in Molecular Biology
Bio 446-546
FALL 2015 M/F 13.00-15.50
Molecular biology of
parasites, snails, plants and fungi
You, Adema, Natvig, Hutchinson
http://biology.unm.edu/cmadema/4546/4546.htm
Today
Introductions
Goals and expectations for this course
Safety video
Organization
Background for research project
Introduction to pipetting
Pipetting exercise
Troubleshooting questions
Introduction
Donald (Don) O Natvig
Professor
(Fungal Genetics)
Coenraad (Coen) M Adema
Associate Professor
(Comparative Immunology/Parasitology)
Miriam Hutchison
PhD Graduate Student
(Fungal Genetics)
NOT “MOLECULAR BIOLOGISTS”, USE RESEARCH TOOLS
WHO ARE YOU?
Molecular Biology
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Tools for biological study
Expensive
Difficult (despite X-files and CSI)
Frustrating
Complex theory hidden in kits, industry
Slow
• Getting bigger BIGGER BIGGER
• Computer intensive BIOINFORMATICS
• Great fun
Goals and expectations for this course
Philosophy and major objective of the Course: It is easy to follow a recipe,
it is harder to follow it well, and most difficult to make it work. Our major
objective is to teach you to get things to work in the lab, this may not be
difficult for some of you, but it may require a personality change for others.
There are 5 identifiable goals in this course:
(1) To introduce you to modern techniques used in molecular biology research.
(2) To teach you how to obtain and apply computer- and internet-based
resources for molecular biology
(3) To teach you to perform research carefully so that your scientific
observations stand on solid ground.
(4) To troubleshoot experiments, but most of all, to persevere when nothing
works.
(5) To give you experience in communicating research findings to the scientific
community.
Ideally, this course will give you a better understanding of what a scientific
career that uses molecular techniques would be like and inspire you to
pursue additional studies.
Syllabus ONLINE
Course information such as lectures, suggested reading, handouts, and
homework assignments will be posted online at:
http://biology.unm.edu/cmadema/4546/4546.htm
Textbook: NOTE there is NO mandatory book for this class.
Readings will be provided through the above website in the form
of protocols, scientific papers, also check out
http://www.ncbi.nlm.nih.gov/sites/entrez?db=Books
For those that prefer the option of a textbook for background
information, we recommend: Molecular Biology of the Gene (7th
ed), Watson, Baker, Bell, Gann, Levine, Losick, Publised by
Pearson student edition ISBN 13:978-0-321-76243-6, ISBN 10: 0321-76243-6
Grading I
Grading (400 points undergrad/500 points for grad students)
Your final grade will depend on:
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midterm examination (take home) (100)
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15 minute class presentation (50)
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your lab book (25)
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Homework/in class troubleshooting exercises (50)
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class attendance/participation (75)
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final paper in journal submission manuscript format (100).
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Additionally, graduate students will be required to give the class a 30 minute presentation on their (proposed)
research (100).
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Unexcused/excessive absences will lower your final grade. Do not make it a habit of arriving to class late. The
class depends on you to be here so that we can start and finish class on time.
FYI-you will work in groups of two/three throughout the semester. You are part of a team, so don’t let your
partners down. Do your share of the work, be respectful of each other’s grasp of the material, help each other
whenever possible. As is common in science, you will collaborate on the writing on your final paper and turn one
paper in per group.
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Warning: There are always slow groups in the class. Please be respectful. It is our experience that these are
often the groups that get the best results..
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Grade Mode Changes: The WP, WF, and WNC are no longer available since Fall semester 2012. Instead, we
have a W (withdrawal) grade mode that does not impact student GPA. (Remember that the course, if dropped
after relevant deadlines remains on the student record and in their tuition account) Like before, this grade mode
does require a Last Date of Attendance (LDA). Please see Fastinfo 3489.
https://unm.custhelp.com/app/answers/detail/a_id/3489/kw/grade%20mode/session/L3RpbWUvMTM0NDg4MDA2Mi9zaWQvS21FTS1CM2w%3D
Grading II
Lab Notebook (25 points):
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You are required to keep a lab notebook for this class. This should be a running commentary with your
ideas and notes to yourself, together with all of the raw data and any calculations you make from the raw
data. Information about the equipment you use should be listed as should information about the
chemicals you use. It should be possible for someone generally familiar with your area of study to pick up
your notebook and repeat or continue your experiments.
All of the exercises will require making calculations or summarizing your results in a notebook. This
should be done during the scheduled lab period for two reasons.
– First, it is usually easier to do calculations when the data is fresh in your mind and you are more
accurate in recalling details for your summaries when you write them immediately.
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Second, the instructor will be available during the lab period and may not be available later. One
of the criteria on which you will be graded is how current you notebook is. Lab notebooks will be
checked by us periodically.
Evaluation: You will be evaluated on the basis of the laboratory notebook you will keep. You should
record everything of interest to you in the lab in this notebook. It is not necessary to reproduce methods
spelled out in the handouts, but everything else should be in your notebook. Not everything has to be
polished, for example, if you want to do a small calculation, do it in your notebook, not on a paper towel
(formerly the record keeping choice of most scientists). Within the last few years society has given clear
signals that it expects scientists to keep reasonable records and we must all learn to do this. All written
communication in the lab will be in this notebook, including feedback from the instructors. Do not write
your raw data on paper towels intending to transfer it to the notebook later. If you want to have a neat
record of your results and feel you cannot do this as the experiment progresses, take the raw data on
one page, and then copy it to a later page in the notebook. You will not be graded on neatness.
Notebook keeping
Lab Notebook:
General Guidelines:
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1. Use a bound book of decent size with numbered pages (hand numbering is okay).
2. Make all entries in ink, beginning with your name and some identification about the notebook.
3. Leave 5 pages or more in the front for a table of contents. For each experiment or set of closely related experiments,
make an entry in the table of contents followed by the date of the experimental work, and the page number where the
research is described in detail.
4. Each experiment should list the
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Title and date
One or two sentences about the experiment
Methods (including calculations and materials)
Summary of the results (generally with some sort of graph or diagram)
Short conclusion stating important findings and/or problems encountered.
5. Changes on previous pages, when needed, should be made by crossing out the old information with one line and
putting the new information nearby. Date any changes made to your notebook.
6. Raw data such as photos of your gels can be cut out and taped into the notebook. When particularly voluminous raw
data is involved, it is best to store this separately, but with a good cross reference system so that the data and the
description of the data can be put together later.
7. Rough graphs can be drawn directly in the notebook; more formal graphs should be drawn on appropriate graph paper
or with a graphing program, and taped into the notebook.
Safety video
Working in the lab
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Laboratory Rules and Guidelines:
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Laboratory etiquette (for your safety):
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No food (this includes chewing gum) or drink is allowed in the lab.
Cell phones, music players and other electronic devices should be turned off.
Books (other than your notebook and lab manual), backpacks, clothing, and personal belongings should not
be kept on lab benches.
Lab coats and protective eyewear are recommended when conducting experiments. Lab gloves should be
worn whenever working with chemicals or solutions. Do not wear shoes with open toes in the lab.
Handle all equipment carefully and don’t goof around in the lab.
Wash and rinse all glassware when you finish using it.
Return individual items to your drawer at the end of each lab.
Notify the instructor if any spills occur or if equipment is damaged, and assist as instructed for the cleanup.
Notify the instructor if anything is spilled on your lab notebook or manual. It is important not to take
contaminated material out of the lab.
Waste disposal:
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Be safe working with dangerous equipment and chemicals,
Ask the instructors when you have questions.
In biohazard bags (tips, tubes, plates, gels)
Sharps disposal:
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Glass and sharp objects should be disposed of in special containers found around the lab. Never, under
any circumstances, put sharp things in the regular trash. This presents a very real hazard for
maintenance workers.
SCHISTOSOMIASIS:A GLOBAL PROBLEM
•207 million humans infected
•Annual mortality 280,000
•Morbidity 120 million humans
•600 million more humans at risk
•Disease impact of schistosomiasis
second only to malaria (WHO)
•Effective chemotherapy, praziquantel currently used in mass treatment
program targeting morbidity by the Schistosomiasis Control Initiative.
•Concern for longevity of parasites, parasite resistance, un-sustainability of
ongoing mass treatment efforts, absence of new drugs and vaccine.
•Continued study of all aspects of schistosomiasis remains valid,
THE LIFE CYCLE OF Schistosoma mansoni
MALE
cercariae
penetrate
human skin
cercariae
released
into water
adult worms
develop in
mesenteric
veins
FEMALE
spined
eggs
passed in
the feces
cercariae
produced
in the snail
digestive
gland
asexual reproduction
occurs in freshwater
snails of the genus
Biomphalaria
miracidium
hatches
from egg in
freshwater,
penetrates snail
http://www.dpd.cdc.gov/DPDx/Default.htm
http://www.biologie.uni-erlangen.de/parasit/contents/research/echino.html
Alaria americanum
Apophallus muehlingi
cal.vet.upenn.edu/merial/Trems/fascio_c.htm
http://parasitology.informatik.uniwuerzburg.de/login/n/h/0103.html
greenmuseum.org/c/vban/trematode.php
Shady lakes facebook
PARASITES AND SNAIL BIOLOGY
DNA
“identity, possibilities”
phylogenetics
RNA
“intentions”
transcriptomics
CTAB/DNAzol
Trizol
gel electrophoresis
nanodrop spec
Bioanalyzer
DNA-free,
PCR
rDNA/mito
TA cloning, B/W screening
electrophoresis
direct sequencing
Sequence ID (BLAST)
editing
Phylogenetics
GenBank
submission
Qiagen plasmid extraction
Restriction digests
M13 sequencing
Primer design, walking
RT-PCR
gel
groups
• You will work in groups of two or three
• Graduate students team up with
UnderGraduates
• groups will be responsible for
– Experimental work
– Final report using experimental data
Time to go through your
drawers
• drawer inventory should match what is in
your drawers
• Mark off items and sign form
• Return to instructors
• We will repeat this on the last day of class
• Name + picture
Pipettors, volume ranges
P20: 2(1)-20 µl = .002-.020 ml (use “yellow” tips)
1
0
= 10.2 µl
2
P200: 20-200 µl = .020-.200 ml (use “yellow" tips)
1
0
= 102 µl
2
P1000: 100-1000 µl = .100-1.0 ml (“blue tips”)
1
0
= 1020 µl (1.02 ml)
2
Pipetting demonstration
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Price of pipettor
Volume
Range, adjust volume
Tips
Double jam, twist (hold the box!)
First stop, pre-wet, view volume, repeat
Dispense: second stop, look for drop or bubble
Mix: down, up and down to first stop
Move tip out of liquid
Eject
Pipetting exercise
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LEARN HOW TO PIPETTE
Tips
Hold the pipettor vertically
Use a smooth up and down motion
Pre-wetting your tip improves accuracy