Lab 0 – Introduction
Waksman Students Scholars Program
Molecular Biology and Genomics Research
(MBGR) in High Schools
2011-2012 Academic Year
Lab Manual
Project Directors:
Dr. Andrew Vershon (NJ) (732) 445-2905
Dr. William Sofer (TX)
Dr. Forrest Spencer (MD)
Dr. Kirby Smith (MD)
vershon@waksman.rutgers.edu
sofer@waksman.rutgers.edu
fspencer@jhmi.edu
KSmith@kennedykrieger.org
Lab Coordinators:
Dr. Janet Mead (NJ)
(732) 445-4661
Dr. Cheryl Warren (MD)
Dr. Stuart Reichler (TX)
mead@waksman.rutgers.edu
cwarren97@yahoo.com
sreichler@mail.utexas.edu
Project Coordinator:
Ms. Sue Coletta (NJ)
coletta@waksman.rutgers.edu
The Lecture Notebook, Lab Manual and Answer Keys to Quizzes and Exams are online at:
WSSP Webpage: http://morgan.rutgers.edu or
WSSP Sakai site https://sakai.rutgers.edu/portal
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Lab 0 – Introduction
Table of Contents
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Laboratory
Laboratory Introduction
A) Project Overview
B) Laboratory Flowchart
C) Clone Naming, School List and numbers
D) General Lab Rules
E) Supplies and Equipment
F) Lab Directory
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1
Pipeting and Determining DNA Concentration
A) Pipeting spotting exercises with P20 (required)
B) Pipeting weighing exercises with P200 and P1000
C) Pipeting weighing exercises with a pipet bulb
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1-10
2
Inoculation of Bacterial Cultures
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3
Setting up PCR Samples
A) Set up PCR reactions
How to program your thermal cycler
Thermal cycler grid sheet
B) Start minipreps
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3-1
3-2
3-3
3-4
4
Agarose gels of PCR Samples
A) Pour Agarose Gel
B) Agarose Gel Electrophoresis
C) How to take a picture of your gel
D) Analysis of the PCR Samples
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4-2
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4-5
5
Plasmid DNA Minipreps
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6
Restriction Digests
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7
Agarose gels
A) Pour Agarose Gel
B) Agarose Gel Electrophoresis
C) Analysis of the Restriction Digest Samples
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7-1
7-3
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Lab 0 – Introduction
A) Overview of the 2011 HiGene project.
The WSSP-11 project is to sequence and
analyze clones from a cDNA library
made from the duckweed Wolffia
australiana. The main goal of the project
is to have every student analyze a DNA
sequence from a clone in the library and
publish the sequence in the GenBank
database at NCBI. This year we are
offering several different options for
involvement in the project to enable
schools with differences in their facilities
and the level of time commitment by the
students and teachers to take part in the
project. A flowchart of the project is
shown with the different laboratory
modules that can be performed in the
analysis of the clones (Fig 1).
Fig 1. Flowchart of the WSSP-11 Project
Students in all of the schools that are part of the project will perform the bioinformatics analysis
of the clones (Lab Module I). Schools that are unable to perform the wet lab experiments will
be provided with DNA sequences for bioinformatics analysis. Schools that have gel
electrophoresis equipment (gel boxes, power supplies, P20, light box and camera) will be given
purified plasmid DNA from clones in the library to perform the restriction digest analysis and/or
PCR (Lab Module II) to determine the size of the insert. Schools with microcentrifuges will be
give plates with the cDNA library so that students can pick and purify their own clones (Lab
Module III). These clones can then be analyzed by restriction digest analysis (Lab Module II).
Schools with a thermocycler and microcentrifuge will pick additional clones from the cDNA
library, screen for the insert size by colony PCR (Lab Module IV), purify plasmid DNA (Lab
Module III), conduct restriction digests (Lab Module II) to determine the size of the inserts and
analyze the sequences. This set of experiments is considered to be the “core project”
Students with clones in which the sequences do not span the entire insert may wish to try
subcloning their plasmids to obtain additional sequence (Lab Module V). If restriction sites for
subcloning are not available, students may wish to design primers to sequence the gap in the
insert (Lab Module VI). Students who find matches to proteins in which the three dimensional
structure has been determined, may want to use molecular modeling programs to examine the
structure (Lab Module VII). Note that only one protein model can be built for each school.
There are several experimental modules that are under development that may be available in the
next year. Once the students finish the bioinformatics analysis of their cDNA clones, they may
want to design primers to PCR amplify a genomic copy of the gene to determine if their cDNA
insert spans intron regions (Lab Module VIII). If it does, students can have the PCR fragment
sequenced to identify the location of the splice point. Students may also want to collect
duckweed samples from local resources and determine the specific species by sequence analysis
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(Lab Module IX). Lab modules V to IX require an advanced understanding of the project along
with more time commitment from the students, teachers and WSSP staff.
B) Overview of the 2011 laboratory experiments.
To screen the Wolffia cDNA library at the
schools, students in schools will 1) Pick
colonies from plates to set up bacterial
overnight cultures; 2) Set up PCR on cultures
and perform the first step of the plasmid
miniprep; 3) Run an agarose gel on the PCR;
4) Complete plasmid minipreps; 5) Set up
restriction digests; and 6) Run agarose gels
on the digests (Fig 2). Depending on the
constraints in time and equipment at each
school, this may take anywhere from 6 to 10
laboratory periods to complete one round of
the screen.
Students may conduct the screen one to four
times. Even if each student at your school is
planning on screening four clones, we
strongly recommend that students only work
on one clone at a time and conduct a round
of the screen four separate times.
Fig 2. Order of experiments for screening for
clones to sequence. Each student should
conduct the screen on one clone at a time.
We suggest that the labs be set up in groups of four students
with each student working through one clone at a time.
Working in groups of four students will allow the students to set up reaction mixes for the PCR
and restriction digests and have an even number of tubes for the centrifugation steps in the
minipreps. This will save time and reagents and will likely reduce mistakes in the procedures.
The laboratory procedures have been written so that they are based on students working on one,
two or four clones at a time.
C) Naming Clones, Gels, and Sequences
i) Clone and Sequences Names:
Each school in the WSSP has been assigned a school number (see table at the right). We will use
the following convention for naming clones: Your school #, your initials, your clone number
and then “.11” for the entire academic year. Use the “.11” even in the spring of 2012.
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Lab 0 – Introduction
For example, if you are from Bayonne (school #01) and
your initials are WS, then your first four clones should be
labeled 01WS1.11, 01WS2.11, 01WS3.11, and 01WS4.11.
If your initials are JP from East Brunswick then your clone
names are 04JP1.11, 04JP2.11, 04JP3.11 and 04JP4.11. If
you have the same initials as someone else at your school,
please include your middle initial (04JSP1.11, etc.).
There are 45 high schools and at least 700 students that
will participate in the WSSP this year. We anticipate that
almost 3000 clones will be isolated, purified, sequenced
and analyzed. Given the large number of schools,
students, and clones that will be in the program, it is
absolutely essential that everyone maintains proper
naming, book keeping, and records for the clones. We are
therefore insisting that everyone adopt a standard
nomenclature (naming method) for their clones, gels and
sequences.
Please do not add dashes or periods in the wrong places,
because it will affect how we search and sort the clones in
the spreadsheets. For example,
01WS1.11 is correct
01-WS1.11 is incorrect
01WS-1.11 is incorrect
01.WS1.11 is incorrect
01WS1-11 is incorrect
01ws1.11 is incorrect
Your DNA sequence files will have the same names as
your clones but there may be a -For and –Rev after the
name to distinguish if they were sequenced with different
primers, i.e 01WS1.11-For and 01WS1.11-Rev will be two
different sequences of the same clones using different
primers.
ii) Naming Gels
Each school will be generating a large number of different
PCR and restriction digest gels. Since multiple students
will run their samples on each gel we have to have a
systematic and efficient method of finding a gel with a
particular student’s samples. The information for each
clone will be entered into a Clone Report Sheet on Google
Docs (Fig. 3).
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School
01. Bayonne HS, NJ
04. East Brunswick HS, NJ
05. High Point HS, NJ
06. Hillsborough HS, NJ
07. James Caldwell HS, NJ
09. JP Stevens HS, NJ
11. Montville HS, NJ
12. New Brunswick HSTHS, NJ
13. Pascack Hills HS, NJ
14. Pascack Valley HS, NJ
15. Rutgers Prep., NJ
16. Somerville HS, NJ
17. The Pingry School, NJ
18. Watchung Hills HS, NJ
19. West Windsor-Plains. HSS, NJ
20. Rutgers University
21. Liberty HS, PA
34. Science & Math Acad. MD
35. Walter Johnson, MD
38. Hackettstown, NJ
40. Bordentown, NJ
49. Piscataway, NJ
50. The Frisch School, NJ
51. Blair Academy, NJ
53. Father Ryan HS, TN
56. Westbury Christian, TX
57. West Morris, NJ
59. Col. Zadok Magruder HS, MD
62. Winston Churchill, MD
64. Rae Kushner Yeshiva HS, NJ
65. Dougherty HS, CA
66. Modesto HS, CA
67. Tracy HS, CA
68. Waipuhu HS, HI
69. Johnathon Dayton HS
70. Old Bridge HS, NJ
71. St. Stephens Episcopal HS, TX
72. Lago Vista HS, TX
73. The Khabele School, TX
74. Dundalk HS, MD
75. Frederick HS, MD
76. Independence School, MD
77. Kenwood, MD
78. Lansdowne HS, MD
79. North Country HS, MD
80. Perrysville HS, MD
81. South River HS, MD
82. Southern HS, MD
83. Arundel Senior HS, MD
84. Woodlawn HS, MD
85. Cherry Hill HS East, NJ
Lab 0 – Introduction
Fig 3. Clone Report Sheet in Google Docs. Proper naming and bookkeeping of the clones,
storage locations and gel images is essential for the analysis of the clones.
Name each gel according to the following convention:
PCR Gels: Will follow the naming convention: School #, PCRG, Gel number, year
(e.g. 20PCRG1.11 for the first gel from school #20; 20PCRG2.11 for the second gel, etc.)
Restriction Digest Gels: Will follow the naming convention: School #, RDG, Gel number,
year
(e.g. 20RDG1.11 for the first gel from school #20; 20RDG2.11 for the second gel, etc.)
NOTE: Since we plan to submit your sequence information to the databases, proper
bookkeeping of your clones and DNA sequence files is absolutely essential!! If we can’t
find it or it is improperly labeled, it will not be submitted for sequencing or submitted to
the database and your work will not get published.
In addition, if we publish the sequence data we are also obligated to provide the clone to
other researchers that are interested in the gene, which will entail finding the correctly
labeled clone. Management of your clones, DNA, report sheets, gel pictures and sequence
files is therefore extremely important!
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Lab 0 – Introduction
D) General Laboratory Rules:
The following are common rules that are found in most university and
industry biology research labs in the United States. The rules for the labs in
your school may differ.
1. Know the location of the laboratory exits.
2. Know the location and use of the safety showers and eye washes.
3. Know the location and use of the fire extinguishers to exit the room.
4. Know the location of the nearest phone that can be used in case of an emergency.
5. Know the potential hazards of the materials, facilities, and equipment with which you will
work.
6. Perform only authorized experiments and follow instructions carefully.
7. Wear proper clothes to protect yourself against chemical spills, dropped objects, etc. This
precludes the wearing of bare midriffs or shorts (unless worn under a lab coat), or sandals.
Confine long hair when in the laboratory.
8. Do not play in the laboratory.
9. Do not work alone in the laboratory.
10. Report all accidents and injuries to a laboratory instructor.
11. No eating, drinking, smoking, applying cosmetics, etc. in the lab.
12. Mechanical pipeting devices are always used. Mouth pipeting is not permitted.
13. Hands should be washed before and after working in the laboratory.
14. Waste must be disposed of properly.
15. Cleaning up: Please clean up after yourself. It is not good scientific practice, and can be
dangerous, to leave things messy. Please be courteous to others who have to use the
equipment and facilities after you.
16. Sterile and stock solutions will be provided by the WSSP. You are responsible for
maintaining the sterility and purity of your solutions. Please notify an your teacher or club
leader if the supplies are getting low or of contamination.
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Lab 0 – Introduction
E) Inventory of Supplies and Equipment:
The WSSP will provide each school with all of the reagents and supplies that you will need to
conduct the experiments for the project. The quantities of each reagent provided to each school is
based on the number of students participating in the project and how many rounds of the screen
that they are going to perform. The quantities of each reagent are in slight excess to account for
small pipeting errors. We may not be able to provide additional reagents if there are repeated
large errors by a school. Please note that most errors are caused by students not having read the
protocols or understand the experiments before they begin the procedure.
You are responsible for the reagents. Make sure that they are properly labeled and stored. That
also includes petri dishes, freezer boxes, cultures, DNA samples, etc.
Equipment required for these experiments is listed in the right column. Equipment will either be
provided by the schools or on loan from the WSSP.
Supplies Provided by WSSP
Equipment supplied by school or
on loan
Lab 1- Pipeting Exercises
Filter Paper
Yellow pipet tips (17/student)
Blue pipet tips (2/student)
10 ml Pipets (1/student)
Weigh boats (1/balance)
Blue Dye (40 ul/students) (1ml)
Microfuge tubes (4/student)
P20 pipetors (>1 per 4 students)
P200 pipetors (>1 per 4 students)
P1000 pipetors (>1 per 4 students)
Pipet bulbs or pump for 10 ml pipets
Electronic balance (optional)
Lab 2- Making ON bacterial cultures
Library Plates (LB+Amp+X-gal)
LB broth + Amp (40 ml)
Sterile culture tubes (1/ON, 25/pk)
10ml pipets
Sterile sticks of picking colonies
Test tube racks
Incubator/shaker (optional)
Refrigerator (store plates and media)
Pipet bulbs or pumps for 10 ml pipets
Lab 3-PCR
yellow tips (5/reaction)
microfuge tubes (1/25/rx)
sterile water in 50ml tubes
Primer-FOR Need 2.5 ul/rx @ 10 pM/ul (100 ul)
Primer REV Need 2.5 ul/rx @ 10 pM/ul (100 ul)
PCR beads -kept dry at room temperature
Labs 4 & 7 - Running Agarose gels
agarose (20 g in 50 ml tube)
Thermal cycler
PCR tube racks (green tip racks)
P20 pipetors (>1 per 4 students)
P200 pipetors (>1 per 4 students)
Agarose gel box, with tray and comb
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Lab 0 – Introduction
weight boats (1/balance, reuse!)
1 kB ladder (20 ul/ gel, 200 ul)
10x loading dye (200 ul aliquots)
EtBr (10 mg/ml) or Carolina Blue
50X TAE buffer (Aliqout of 40 ml)
Roller bottles
Latex Gloves(L/M/S) X-Large
Funnel for recycling the running buffer
Power supply
Microwave
UV box and digital camera
P20 pipetors (>1 per 4 students)
Graduate cylinder
250 ml gel for melting agarose gel
Microfuge tube racks
Weigh balance
Lab 5 - Minipreps
Microfuge tubes (2/prep)
Blue pipet tips ( 4/prep if pour)
Yellow pipet tips (1/prep)
miniprep solution I (0.2ml/Rx - aliquot 6 ml)
miniprep solution II (0.2ml/Rx - aliquot 6 ml)
miniprep solution III (0.2ml/Rx - aliquot 6 ml)
miniprep solution Wash (0.4ml/Rx - aliquot 12 ml)
miniprep solution E (0.5 ml water)
spin column
collection tubes
storage boxes (2 each/school)
15 ml tubes for buffers
Lab 6 - Setting up Restriction Digests
Buffer 4 (10x) (2.5 ul/rx) (100 ul)
PvuII enzyme 1.25 ul/RX (Variable)
Yellow tips (4/rx)
Microfuge tubes ( 1.25/rx)
Microcentrifuges
Microfuge tube racks
P200 pipetors (>1 per 4 students)
P1000 pipetors (>1 per 4 students)
P20 pipetors (>1 per 4 students)
P200 pipetors (>1 per 4 students)
37oC Temperature block or water bath
Microfuge tube racks
Freezer (store enzyme and DNA samples)
F) Lab Directory:
Balance: Weighing balances will be used to weigh out agarose and other chemicals. Weigh
boats and spatulas can be found in the drawer below the balances. Please recap all bottles, clean
up your spills, and keep the balance area clean.
Camera: The camera set-ups are used to record a picture of your agarose gels. Images of these
gels will be mocked up and uploaded to your school’s GoogleDocs site. The UV light from the
light box can burn your eyes. Always use glasses and/or the protective shield when viewing
your gels. Clean up after yourself when you are done with the camera.
Centrifuges: Always balance the centrifuges. Make sure you balance opposing buckets of the
centrifuges and that your tubes have approximately the same volume. Upon starting your
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centrifuge spin, stay with the centrifuge until it has reached the speed that you set. Immediately
turn off the centrifuge if there are any strange noises. If the centrifuge is excessively noisy,
check whether your tubes are balanced.
Electrophoresis: The high voltage used during electrophoresis constitutes a serious hazard.
Always turn off the power supply before touching the agarose gel boxes, and always keep the
box, leads and power supply away from the edge of the bench. Improper use can be dangerous!
Make sure your power supply is turned off before connecting and disconnecting gel boxes.
Enzymes: All restriction enzymes should be stored in the freezer and on an ice bucket when you
are working to set up the reaction.
i) Bring you samples over the to the ice bucket with your P-20 pipet properly set to the
correct amount of enzyme you will need.
ii) Many of these enzymes are very labile and quite costly. Hold the enzyme by the side of
the tube and not by the bottom to not warm the enzyme. Negligence on your part could
destroy everyone's experiments.
iii) ALWAYS USE A NEW TIP! Cross-contaminating enzymes or contaminating them
with DNA will also ruin everyone's experiments.
Ethidium Bromide: Some schools may use Ethidium bromide to visualize DNA under UV
light. This chemical is a strong mutagen. Minimize your contact with it and always wear gloves
whenever handling it.
Freezers: A freezer is required to store the PvuII enzyme, 10X buffer, primers, and 1 KB
ladder and your plasmid DNA minipreps. DO NOT store the cDNA library plates or the
LB+Amp media in the freezer.
Incubators: The incubators in the lab are to remain at the preset temperatures. One incubator
will be set at 37˚C for growing bacteria and perform your DNA digests.
Microwave Ovens: Microwave ovens are used in the lab for melting agarose for your DNA
gels. Place the bottle with your agarose gel solution in the machine and set the timer. BE SURE
THE CAP OF THE BOTTLE IS OFF OR VERY LOOSE! The two greatest potential hazards
are a steam explosion from heating tightly sealed containers or superheated liquids that boil over
when you take the bottle out of the machine. You can avoid superheating by heating your
solution in short bursts. Use caution when removing the bottles and wear gloves and safety
glasses. Make sure there is no metal (including aluminum foil) in the microwave. Do not use
any of the lab microwave ovens for heating food.
Pipetmen: Pipetmen are used for measuring and delivering very small liquid volumes in the
range of 1-1000 µl. They generally come in three sizes P-20, P-200 and P-1000 pipetman for the
course. You will use these instruments more than any other in the project so please take good
care of them. They are delicate and cost about $250 each. If you notice that your pipetman is
leaking or does not draw properly notify your teacher. Pipetmen also present a major source of
contamination to DNA samples, restriction enzymes, etc. because they can aerosolize the liquid
you are measuring. Therefore try to draw the liquid up slowly. (See Lab 1 for a more complete
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description).
Pipets: We will use individually wrapped sterile plastic serological pipets for measuring liquid
media and solutions. These pipets cannot be reused. If they come in contact with a bench or
your hand they are no longer sterile and should be discarded.
Power Supplies: A low voltage power supply is used for running agarose gels. Keep the power
supply and gel box away from the edge of the bench in a spot where you are not constantly
reaching over it or bumping it as you do your other work. It is wise to always turn the power off
before handling the gel box. Never disconnect the leads and leave them on the bench with the
power supply on.
Pipet Bulbs: A black pipet bulb for pipeting volumes of liquid between 1-25 ml. No mouth
pipeting is allowed in the labs.
Reagents: In order to make the project run smoothly, you will be provided with all of the
reagents that you will need to do your experiments. You are responsible for your own reagents
at your bench. Make sure that they are properly labeled. That also includes petri dishes, freezer
boxes, cultures, DNA samples, etc. Before you start an experiment check to see that you have
enough of the reagents on hand.
Refrigerators: Are used to store petri plates containing the cDNA library and LB+ Amp media.
Label and date everything that you put in the refrigerator.
Sterile reagents: All biological materials or things that may have come in contact with
biological materials should be kept sterile to prevent contamination. This includes: pipet tips,
microcentrifuge tubes, culture tubes, LB+Amp media, 10 ml pipets and many solutions. The
best way to keep items sterile is to reduce exposure to open air. This includes not leaving the
tops off the pipet tip boxes for extended periods of time, tube bags open, unwrapped 10 ml pipets
on the bench before using, and caps off the culture tubes. If you think a tip or pipet touched the
bench then discard it. Do not hold microfuge tubes by the cap. Always check the LB media
for growth before you use it!
UV Transilluminator: This piece of equipment is used with the camera set-up to visualize
DNA and to take pictures of your agarose gels. WEAR PROTECTIVE GLASSES. Prolonged
exposure to skin can cause severe burning.
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