Kazakh National Agrarian University
Faculty: Technology and bio resources
Department: Technology of producing livestock
products
Biotechnology
Size Exclusion
Chromatography
Antibodies and their applications
Cells as “Nanofactories”
Genes in a bottle kit
SDS page
Course for Master students
Author: PhD biological science, associative
professor Elizaveta Kan
2013
To the Teacher
One of the biggest challenges for those studying biotechnology for the first time is that
many of the events and processes they are studying are invisible. Bio-Rad's Explorer products
offer a unique solution. All of our educational kits use colored or fluorescent molecules so
that the biological processes that are being studied can be clearly and easily visualized.
Ths Size Exclusion Chromatography (SEC) kit is designed to teach basic gel
filtration chromatography techniques. This kit utilizes the colored molecules hemoglobin
and vitamin B12 to illustrate the principles of SEC. Students can easily visualize the
separation of these molecules as they pass through the chromatography column.
A Complete Teaching Guide
Developed over five years, Biotechnology Explorer kits and curricula have been
written for teachers, by teachers, and have been extensively field-tested in a broad range
of class-room settings from high school through the undergraduate level. Easy-to-use
Biotechnology Explorer kits are the perfect way to bring the excitement of biotechnology
into the classroom. Each kit contains an innovative step-by-step protocol, which makes
the kits the perfect choice for both experts and beginning teachers.
The curriculum contained within the manual for each kit makes our products unique.
Each kit contains its own unique curriculum package which is divided into a Teachers Guide
and Student Manual. The Teachers Guide is divided into three sections. One section contains
background information, lecture topics, and suggested references which will enable each
teach-er, both the experienced and the newcomer to biotechnology, to prepare and design
lectures and lessons which can precede the actual labs. This advance preparation will virtually
insure that the labs run smoothly and that the students will understand the concepts behind
each lab. There is a detailed section on the laboratory set up, complete with simple procedures
which contain graphic diagrams detailing the advance preparation for the labs. This makes the
set up for each lab simple and straightforward. In addition, this section contains time tables
which will help you plan your schedule. Each lab can be performed in a 50 minute class
period, which should fit into most schedules.
Finally, we provide a detailed Teachers Answer Guide which contain answers to all of
the questions posed in the Student Manual. The teacher can use these answers as a guide
when reviewing or grading the questions presented in the student section of the manual.
The Student Manual is designed to maximize student involvement in both the labs
and the thought questions embedded in each lesson. Student involvement in this process
will result in an increased understanding of the scientific process and the value of
proceeding into a task in an organized and logical fashion. Students who engage in the
science curriculum found in the Bio-Rad explorer kits develop a positive sense of their
ability to understand the scientif-ic method.
We strive to continually improve our curriculum and products. Your input is
extremely important to us. Incorporation of your ideas, comments, critiques, and
suggestions will enable the Explorer products to evolve into even better teaching aids.
You can find the catalog and curriculum on the internet. Look up our home page at
www.bio-rad.com or call us at 1-800-424-6723.
Ron Mardigian
Director, Biotechnology Explorer
Program ron_mardigian@bio-rad.com
Table of Contents
Page
Instructors Guide
Kit Inventory Checklist .................................................................................. 2
Implementation Timeline ............................................................................... 3
Introduction to Chromatography ...................................................................... 4
Principles of SEC .................................................................................... 5
The Sample—Hemoglobin and Vitamin B12 ................................................ 6
Workstation Checklist ................................................................................... 8
Advance Laboratory Preparation...................................................................... 9
Instructors Lab Manual .................................................................................10
Laboratory Quick Guide: Graphic Laboratory Protocol .......................................13
Student Manual
Lesson 1
Lesson 2
Introduction to Chromatography .................................................15
The Sample ............................................................................18
Chromatography Laboratory ......................................................21
Lesson 3
Analysis of Laboratory Result ....................................................25
Appendices
Appendix A Teachers Answer Guide ............................................................26
Appendix B
Glossary of Terms ....................................................................30
ii
Student Objectives
•
Compare and contrast the use of different types of column chromatography in the
purifi-cation of proteins.
•
Explain how naturally occurring or recombinant proteins are separated and purified
using column chromatography.
•
Discuss how the structure and biochemical properties of proteins relate to
purification using column chromatography.
•
Apply the scientific method to solve a problem*
* Problem: Can Hemoglobin (molecular weight of 65,000 daltons) be separated from
vitamin B12 (molecular weight of 1,350 dalton) by gel filtration chromatography?
Pre-Lab Activities
The following activities are recommened before chromatography is conducted:
1. Cover Biology text on protein structure.
2. Review DNA structure and function and protein synthesis.
3. Conduct library and online research studying the functions of some common proteins.
Background Lectures Ideas
Our bodies contain thousands of different proteins which perform many different
jobs. Digestive enzymes are proteins; some of the hormone signals that run through our
bodies and the antibodies protecting us from disease are proteins. The information for
assembling a pro-tein is carried (in code) in our DNA. The section of DNA which
contains the code for mak-ing a protein is called a gene. There are thousands of genes on
each chromosome. Each gene codes for a unique protein. The gene which makes a
digestive enzyme in your mouth is dif-ferent from one which makes an antibody.
Proteins are often products sought to be used for medical purposes. Some of these
proteins are purified in large quantities from a naturally-occurring source. Recently,
many proteins for medical purposes have been made through genetic engineering and
recombinant DNA technology. No matter what the source, a protein of interest is found
in a mixture of a cell’s other proteins. Some cells, such as bacteria, produce large
quantities of up to two thousand dif-ferent kinds of proteins.
Since 75% of the dry matter in living things is protein, biologists must often purify a protein of interest from other proteins in a cell. Determining the procedures for the purification
of a particular protein is a challenging task for the biotechnology industry. To separate any of
the macromolecules, scientists utilize their knowledge of the chemistry of these molecules,
including: the molecular weight of the protein (size), its charge, and its shape.
1
Instructors Guide
Kit Inventory Check (✔) List
This section lists the components provided in the Size Exclusion Chromatography kit. It also
lists required accessories. Each kit contains sufficient materials to outfit eight student workstations. Use this as a checklist to inventory your supplies before beginning the experiments.
Kit Components
Number/Kit
(✔)
Protein Mix
Hemoglobin
Vitamin B12
Poly-Prep® sizing columns
1 vial
❏
8
❏
**
Column end-caps
Column buffer
Pipettes (1 ml)
Collection tubes
Manual and Quick Guide
**
25
50 ml
10
100
1
❏
❏
❏
❏
❏
8
8
❏
❏
Several extra are supplied with the kit.
Required Accessories
Test tube rack for holding 12 tubes
Black marking pen
2
Implementation Timeline
The active lab session is designed to be carried out in a single 50 minute period. The
detailed laboratory protocol can be found in the Student Manual.
Lesson 1
Lesson on Chromatography
Lesson on hemoglobin, RBCs, vitamin B12, protein biochemistry.
Analysis and thought questions.
Lesson 2
Run the Laboratory
Lesson 3
Analysis of results
Analysis questions
3
Lesson 1
Introduction to Chromatography
This investigation is intended to teach basic techniques of size exclusion
chromatography. This laboratory activity integrates well into both basic and advanced
biology curricula. The two molecules used in this activity, hemoglobin and vitamin B12,
are both compounds essen-tial to functions in the human body; thus this laboratory
activity can be linked to basic lessons in biology, human physiology, and biochemistry.
This section describes the experimental and conceptual points which may prove challenging to students. These points are extremely important to the overall outcome of the
activ-ity. Instructors should direct their students attention to these points, and when
possible, demonstrate the technique before the students attempt the procedure.
Chromatography is commonly used in biotechnology for purifying biological molecules, like
proteins, for medicine or other uses. Chromatography separates individual components from
complex mixtures. Chromatography consists of a mobile phase (solvent and the molecules to be
separated) and a stationary phase either, in paper (in paper chromatography) or glass beads, called
resin, (in column chromatography), through which the mobile phase travels. Molecules travel
through the stationary phase at different rates because of their chemistry.
Some Common Types of Chromatography
In gel filtration chromatography, commonly referred to as size exclusion
chromatog-raphy (SEC), microscopic beads which contain tiny holes are packed into a
column. When a mixture of molecules is dissolved in a liquid and then applied to a
chromatography column that contains porous beads, large molecules pass quickly around
the beads, whereas smaller molecules enter the tiny holes in the beads and pass through
the column more slowly. Depending on the molecules, proteins may be separated, based
on their size alone, and frac-tions containing the isolated proteins can be collected.
In affinity chromatography, a biomolecule (often an antibody) that will bind to the
pro-tein to be purified is attached to the beads. A mixture of proteins is added to the
column and everything passes through except the protein of interest, which binds to the
antibody and is retained on the solid support. To get the protein to elute from the column,
another buffer is used to disrupt the bond between the protein of interest and the
antibody. Often this elution buffer contains high concentrations of salt or acid.
In ion exchange chromatography, the glass beads of the column have a charge on them
(either + or -). A mixture of protein is added to the column and everything passes through except
the protein of interest. This is because the charge of the beads is picked to have the opposite charge
of the protein of interest. If the charge of the beads is positive, it will bind neg-atively charged
molecules. This technique is called anion exchange. If the beads are negatively charged, they bind
positively charged molecules (cation exchange). Thus, a scientist picks the resin to be used based
on the properties of the protein of interest. During the chromatography, the protein binds to the
oppositely charged beads. After the contaminant is separated from the protein of interest, a high
salt buffer is used to get the desired protein to elute from the column.
This kit is designed to teach basic principles of size exclusion chromatography (SEC), a
technique which allows the separation of molecules on the basis of size. The kit uses the colored molecules hemoglobin and vitamin B12 to illustrate the principles of SEC. Hemoglobin
(reddish-brown) is much larger than vitamin B12 (pink), and thus passes through the column
more quickly than vitamin B12. The students can easily visualize the separation of these
molecules as they pass through the column and into collection tubes.
4
Principles of Size Exclusion Chromatography (SEC)
The mass of beads within the column is often referred to as the column bed. The
beads act as “traps” or “sieves” and function to filter small molecules which become
temporarily trapped within the pores. Larger molecules pass around, or are “excluded”
from, the beads. This kit contains eight columns which are prefilled with beads that
effectively separate or “fractionate” molecules that are below 60,000 daltons. As the
liquid flows through the col-umn, molecules below 60,000 daltons enter the beads and
pass through the column more slowly. The smaller the molecules, the slower they move
through the column. Molecules greater than 60,000 pass around the beads and are
excluded from the column—also referred to as the exclusion limit of a column.
The liquid used to dissolve the biomolecules to make the mobile phase is usually called a
buffer. The mixture of biomolecules dissolved in the buffer is called the sample. The sample is placed on the column bed and the biomolecules within the buffer enter the top of the
col-umn bed, filter through and around the porous beads, and ultimately pass through a small
opening at the bottom of the column. For this process to be completed additional buffer is
placed on the column bed after the sample has entered the bed. The mobile phase liquid is
col-lected, as drops, into collection tubes which are sequentially ordered. A set number of
drops is usually collected into each tube. The larger molecules which pass quickly through the
col-umn will end up in the early tubes or “fractions”. The smaller molecules which penetrate
the pores of the stationary phase end up in the later fractions.
Hemoglobin and vitamin B12 are the two molecules which are being separated in
this lab activity. Hemoglobin, which is brown, has a molecular weight of 65,000 daltons
and is thus excluded from the column. Hemoglobin will pass more quickly through the
column and appear in the early collection tubes, or fractions. Vitamin B12, which is
pink, has a molecu-lar weight of 1,350 daltons and is thus fractionated by the column.
The vitamin B12 molecules penetrate the pores of the beads, becoming temporarily
trapped. As a result, they pass much more slowly through the column and appear in the
later fractions. The schemat-ic below illustrates the differential fractionation of large
and small molecules on a size exclu-sion column.
A mixture of large
and small proteins
is applied to a column of porous
beads.
As the buffer flows
down the column,
the small protein
molecules penetrate
into the beads and
are slowed.
Fraction 1
The larger protein
molecules
emerge from the
column first.
Fraction 2
Fraction 3
5
The Sample—Hemoglobin and Vitamin B12
Hemoglobin
Hemoglobin, a protein found in red blood cells, functions to transport oxygen to the tissues of the body. The hemoglobin used in this experiment is bovine hemoglobin. The use of
bovine hemoglobin (rather than the human counterpart) avoids the potential health hazard
presented when using human blood products. Hemoglobin is made up of four polypeptides
(small proteins) which associate to form a large, globular protein. Hemoglobin gets its name
from the heme group, the iron-containing component of hemoglobin which physically binds
oxygen. The iron-containing heme group is responsible for the red-brown color of
hemoglobin. The closely related protein, myoglobin, is found in muscle and is responsible for
delivering oxygen to muscle tissue. Muscles which are very active and require a lot of oxygen
are dark in color because of a high myoglobin content. An example would be the red-brown
color of the dark meat of chicken.
Hemoglobin is the main component of red blood cells (RBCs), the oxygen carrying cells of the
body. Again, it is the heme group of hemoglobin which gives RBCs their distinctive red color.
Different forms of hemoglobin are produced during different stages of development. Fetuses
produce a form of hemoglobin which has a higher affinity (tighter binding) for oxy-gen than does
adult hemoglobin. Because fetuses depend upon their mothers for their oxygen supply, it is
important that maternal hemoglobin can easily give up its oxygen to the fetal hemoglobin. For this
reason, obstetricians advise their patients to avoid vigorous exercise during pregnancy. Vigorous
exercise depletes the tissues of oxygen, which sets up a compe-tition between the transfer of
oxygen to maternal tissues or to fetal hemoglobin.
In addition to oxygen, hemoglobin can also bind carbon monoxide. Hemoglobin
actual-ly has a higher affinity for carbon monoxide than for oxygen. Suffocation from
carbon monox-ide occurs when oxygen bound to hemoglobin is displaced by carbon
monoxide, which in turn deprives body tissues of oxygen.
The body can adapt to environmental changes which require increased amounts of
oxy-gen delivery to tissues. At high altitudes, where the amount of oxygen in air is
decreased, the body responds by increasing the number of red blood cells produced. This
effectively increas-es the number of molecules of hemoglobin in the blood supply, which
has the effect of increas-ing the oxygen supply to the tissues. For this reason, athletes
will train at high elevation to increase the amount of RBC, and thus increase their
oxygen capacity, which is needed for rigorous exercise.
Sickle cell anemia is a molecular disease of hemoglobin. A single change or mutation in
the gene which encodes hemoglobin results in a mutation in the amino acid sequence. This
mutation changes the three dimensional structure of the polypeptides of hemoglobin, causing
them to “stick” together as rod-like structures. The abnormal rod-like hemoglobin molecules
distort the structure of red blood cells, causing them to have a sickle shape. Unlike their round
counterparts, the sickle-shaped RBC can not freely pass through capillary beds, and thus the
capillary beds become blocked. The blocked capillary beds of organs and tissues make delivery of oxygen difficult, resulting in extreme fatigue and even death. Because sickle-cell anemia is a genetic disorder which results from a mutated genetic sequence, at this time there is
no cure. However, the side effects of sickle cell anemia can be alleviated by frequent blood
transfusions from people who have normal hemoglobin and red blood cells. Sickle cell anemia is a genetic disease in which the individual has inherited a defective mutant hemoglobin
gene from both parents. Individuals with the sickle cell trait have received an abnormal gene
from only one parent, and the single defect actually confers an evolutionary advantage. In
Africa, expression of the sickle cell gene positively correlates with malaria infections. Malaria
is a deadly disease caused by a mosquito-borne parasite. The parasite infects and ultimately
6
kills RBCs. The parasite can infect normal RBCs, but can not infect sickle cell RBCs. Thus,
the sickle cell trait helps confer resistance to malaria and results in a positive evolutionary
adaptation. Unfortunately, expression of two copies of the gene is deleterious.
Vitamin B12
Vitamin B12 is a vitamin that is essential to humans and other vertebrates. Vitamin
B12 is an essential cofactor of several biochemical reactions which occur in the human
body. One function of vitamin B12 is the breakdown of fats. Sources rich in vitamin B12
include eggs, dairy products, and meats. Vitamin B12 is not found in plants and
vegetable foods. Thus people who have strict vegetarian diets are often deficient in
vitamin B12, unless they take some supplementary vitamin.
Pure molecules of vitamin B12 can not be absorbed by the intestines. Vitamin B12
must bind to a carrier protein in the intestinal tract. When vitamin B12 binds to this
carrier pro-tein, the complex is able to pass through the intestine and into the blood
stream, where it is eventually taken up by the liver.
Because vitamin B12 is only required in minute quantities (humans require ~3 µg/day),
vitamin B12 deficiencies are extremely rare. However, some individuals have a genetic disorder in which the gene that codes for the carrier protein is mutated. Individuals with this
mutation do not synthesize the carrier protein necessary for absorption into the blood stream.
Thus, even though these people have adequate intakes of vitamin B12, they still show signs of
deficiency because they lack the required carrier protein.
7
Laboratory Workstation (✔) Checklist
Student Workstations. Materials and supplies that should be present at each student
workstation prior to beginning each lab experiment are listed below. The components
provided in this kit are sufficient for 8 student workstations.
Instructors (Common) Workstation. A list of materials, supplies, and equipment
that should be present at a common location that can be accessed by all student groups is
also list-ed below. It is up to the discretion of the teacher as to whether students should
access com-mon solutions, or whether the teacher should aliquot solutions.
Student workstation items
Number required
(✔)
Collection tubes
Size exclusion chromatography columns
Column end-caps
Pipette
Lab marker
Test tube rack
12
1
1
1
1
1
❏
❏
❏
❏
❏
❏
Instructor workstation items
Number required
Protein mixture
Column buffer
1 vial
1 bottle
8
❏
❏
Advance Laboratory Preparation
This section describes the preparation that needs to be performed by the instructor
before the laboratory. An estimation of preparation time is included in each section.
Advance Preparation
Objectives
Rehydrate protein mixture
Set up student and instructor workstations
Photocopy Quick Guides for students
Time required Twenty minutes to 1 hour
Procedure
Approximately 15 minutes before the start of the laboratory, use one of the
pipettes in the kit and add 0.5 ml of distilled water to the vial of protein
mix. Mix gently several times over the course of 15 minutes. Keep on ice
or in the refrigerator until the start of the experiment.
9
Lesson 2 Laboratory
Instructors Lab Manual
This version of the lab protocol contains detailed notes and helpful hints for setting
up and running the lab.
Techniques to Highlight
Pipetting
Before beginning the experiment, point out to the students the graduation marks on the
pipet. The 250 µl and 1 ml marks will be used for measurements in this exercise. Have the
stu-dents practice with volumes of water to acquaint themselves to precision pipetting.
1 ml
750 µl
500 µl
250 µl
100 µl
Chromatography
Also stress that it is important not to disturb the column bed. When loading sample
or buffer onto the column bed, the pipette should be inserted close to the bed against the
wall of the column. Liquid should be gently expelled from the pipette down the wall of
the column (for the buffer) or onto the top of the bed (for the protein mix).
Buffer
Protein mix
Important hints for successful chromatography
1. Snap, do not twist, the bottom tab from the prefilled column.
2. Place the column gently into the collection tubes. Jamming the column tightly into the
col-lection tubes will create an air tight seal and the sample will not flow through. You
can create a "paper crutch" by folding a small piece of paper, about the size of a match
stick, and wedging it between the column and the collection tube. This crutch makes it
impos-sible for an air tight seal to form, and insures that the column will flow.
3. The columns are designed to drip slowly. The entire chromatography procedure should
take 20 to 30 minutes. It is important not to remove the column more than needed from
collection to collection tube, as motion can cause major disturbance to the column bed.
10
Setting Up and Running the Lab
1. Each student team will require 12 collection tubes. Have each team label 10
collection tubes sequentially from 1 to 10. The last two tubes are labeled “waste” and
“column buffer”. Place the tubes in the rack. Label either the tubes or the rack with
your name and laboratory period.
2. Pipet 4 ml of Column Buffer into the tube labeled column buffer. There is only one
stock bottle of column buffer provided in this kit. The teacher may aliquot the 4 ml
into each of the labeled collection tubes, or one student from each group may aliquot
their own 4 ml of column buffer.
3. Have the students remove the top cap and snap off the end of their Poly-Prep sizing col-
umn. Drain the buffer into the “waste” collection tube. Then recap the bottom of the
col-umn with the column end cap.
4. Place the column onto tube 1. The students are now ready to load (or the teacher may
choose to load) the protein sample onto the column. There is one vial of protein mix
in the kit—it may be most convenient to approach individual student groups with the
vial and load a drop onto the column.
5. Have students remove the end cap from the column. Observe the top of the column bed;
all of the buffer should have drained from the column. This is best observed by looking
directly over the column—the “grainy” appearance of the column beads should be visible. If any residual buffer remains on top of the column, the protein sample will be diluted when a drop is applied, which will result in poor separation. Carefully load one drop
of protein mix onto the top of the column bed. The pipette should be inserted into the
column and the drop should be loaded just above the top of the column bed so that application of the protein sample minimally disturbs the column bed.
6. Allow the protein mix to enter the column bed. This is best observed by looking
directly over the column. Then, carefully add 250 µl of column buffer to the top of
the column. This is best done by inserting the pipet tip into the column so that it rests
just above the column bed. Carefully let the buffer run down the side of the tube and
onto the top of the bed. Begin to collect drops into tube #1.
7. When all of the liquid has drained from the column, add another 250 µl of column
buffer to the top of the column. Add the buffer as before, by placing the pipette just
above the top of the column and letting the buffer run down the side of the tube.
Continue to collect drops into tube 1. The number of drops that are collected into
tube 1 do not need to be counted.
8. When all of the liquid has drained from the column, add 3 ml of column buffer to the top
of the column. This can be done by adding 1 ml from the pipette three times. At this time
the protein mix has entered the column far enough so that slight disturbances to the column bed will not affect the separation. Transfer the column to tube 2 and begin to count
the drops that enter into each tube. Collect 5 drops of buffer into tube 2. Collect 5 drops
into each tube, with the exception of tube 10, into which 10 drops will be collected. The
teacher can point out that as one student loads the column, another student can count the
drops as they drop into the collection tubes.
9. When 5 drops have been collected into tube 2, transfer the column onto tube 3.
Collect 5 drops of buffer into each collection tube. When 5 drops have been collected
into a tube, lift it off and transfer it to the next tube.
10. Continue collecting 5 drops into each tube. When you reach tube 10, collect a total of
10 drops. After the last 10 drops have been collected, cap the column.
11
11. The collection tubes containing the column fractions can be parafilmed or covered
and stored in the refrigerator. If tightly sealed, the fractions can be stored for ~ 1
week for future observations/discussions. The column can also be capped with the
top and end caps and stored in the refrigerator for ~ 1 week.
12. It may be interesting for the students to compare the starting mix with their
individual frac-tions. You can take the remaining column buffer and add ~ 5 drops of
protein mix to the bottle. You can then aliquot 5 drops of this “starting mix” into
each of the students “waste” tube. The student groups can then compare the starting
mixture with the size-fractionat-ed samples.
Antibodies and their applications
Poly- and Monoclonal Antibodies
Poly- and Monoclonal Antibodies
Poly- and Monoclonal Antibodies
Hybridoma technique
Monoclonal antibodies production
Cryoconservation of Hybridomas
Structure of Antibodies
Structure of Antibodies
Immunoglobulins
Basic structure
• heavy and light chains
• disulfide bonds
• variable and constant regions
• hinge region
• domains
• carbohydrates
Immunoglobulins
Classes: structure & properties
• somatic recombination large variety of immunoglobulins
• light chains genes 3 segments V (variable)
J (joining)
C (constant)
300
4
1
• heavy chains genes 4 segments V (variable)
D (diversity)
J (joining)
C (constant)
500
12
4
5
Immunoglobulins
Classes: structure & properties
IgG
IgM
IgA
IgD
IgE
2. Immunoglobulins
4. classes: structure & properties
IgG • serum(75%)/extra vascular IgM
spaces
• placental transfer
• binding to cells
IgA
IgD
IgE
2. Immunoglobulins
2.4 classes: structure & properties
IgG • serum(75%)/extra vascular
spaces
• placental transfer
• binding to cells
IgM • frequency differs
• first produced by
fetus & after first
antigen-contact
• good agglutinating
IgA
IgD
IgE
2. Immunoglobulins
2.4 classes: structure & properties
IgG • serum(75%)/extra vascular
spaces
• placental transfer
• binding to cells
IgM • frequency differs
• first produced by
fetus & after first
antigen-contact
• good agglutinating
IgA • serum/secretories
• major class in secretions
 important in local immunity 
IgD
IgE
2. Immunoglobulins
2.4 classes: structure & properties
IgG • serum(75%)/extra vascular
spaces
• placental transfer
• binding to cells
IgM • frequency differs
• first produced by
fetus & after first
antigen-contact
• good agglutinating
IgA • serum/secretories
• major class in secretions
 important in local immunity 
IgD • serum(minimal)/B cell
surfaces
• receptor for antigen on
B cell surfaces
IgE
2. Immunoglobulins
2.4 classes: structure & properties
IgG • serum(75%)/extra vascular
spaces
• placental transfer
• binding to cells
IgM • frequency differs
• first produced by
fetus & after first
antigen-contact
• good agglutinating
IgA • serum/secretories
• major class in secretions
 important in local immunity 
IgD • serum(minimal)/B cell
surfaces
• receptor for antigen on
B cell surfaces
IgE • Fc-receptors on basophils/mast cells
• allergic reactions
Immunohistochemistry
Human
carcinoma of the
bladder (mTOR Antibody)
Human
prostate carcinoma
(Cox IV Antibody)
Immunofluorescence
Endothelial cells (Actin, Nuclei)
Human cervical cancer
(β-catenin, Actin, Nuclei)
Skin fibroblasts (Mytochondria,
Filamentous Actin, Nuclei)
Intracellular structure of HeLa cells
(Nucleus, Golgi, Microtubules)
Flow Cytometry
Western blotting
Band pattern interpretation
Lane 1, HIV + serum
(positive control)
Lane 2, HIV - serum
(negative control)
Lane A, Patient A
Lane B, Patient B
Lane C, Patient C
IL-4 + anti-STAT6
unstim.
IL-4
IL-4
IL-4 + anti-STAT6
unstim.
IL-4 + anti-STAT6
unstim.
IL-4
αIFNαIFN- + antiSTAT1
αIFN- + antiSTAT5
αIFN- + antiSTAT6
unstim.
Electrophoretic-Mobility-Shift Assay
Cell line
G-401
SK-NEP-1
WT 3a/b
Interferon-α
-
STAT1
STAT1
Interferon-γ
STAT1
STAT1
STAT1
IL-4
STAT6
STAT6
STAT6
IL-6
STAT3
STAT3
STAT3
HGF
-
-
-
SCF
-
-
-
NFκb
NFκb
NFκb
GF, Cytokine
Supershift
Supershift
STAT 6:6
STAT 5:6
STAT 5:5
STAT 1:1
Daudi
(control cells)
G-401
SK-NEP WT3a/b
TNF-a
Max Wilms
1867-1918
Wilms Tumor (I)
• Solid embryonic tumor of the
kidney (incidence 1 in 10‘000)
• Different histological subtypes
• Different stages (SIOP protocol)
• 90% unilateral, 10% bilateral
• 10-15% unfavourable outcome (histology, stage)
Wilms Tumor (II)
before resection
after resection
Wilms tumor cell lines
T
U
M
O
R
CK8
CK18
Vimentin
N
O
R
M
A
L
Immunofluorescence staining
Wilms Tumor Cell Lines
G-401 (ATCC, p.52)
•
•
•
•
•
•
•
Established from 3 m old
baby (46, XY karyotype)
Morphology: epithelial
Rhabdoid tumor?
SK-NEP-1 (ATCC, p.38)
Established from 28 y.
old patient
Hypotriploid to hypertriploid
karyotype
Morphology: epithelial like
Clear cell sarcoma?
•
•
•
WT 3a/b (Dr. Stock)
Established from 5- y old boy
Heterogeneous complex
karyotypes
Growths in epithelial clusters
Nubia cell line (UKBB)
•
•
•
Established from 2- y old girl
Normal karyotype (46, XX)
Growths in epithelial clusters
G-401 cell line
12
FISH: cep 12 + wcp 7
der 12
7
7
7p22
?
?
?
G-401cell line
FISH: wcp 12 + 7p22
pathological Metaphases
7
7p22
der 12
12
7
G-401 (normal and pathological metaphases)
120
100
80
pathological
60
normal
40
20
0
2
4
7
10
passage number
12
15
Proliferation assay with the clones of G401 cell line
60
60
50
50
40
40
day 3
day
30
1B11
5
day 7
2E8
30
1E8
day 10
20
3D5
20
10
10
0
1B11
2E8
1E8
clones
•
•
•
3D5
0
3
5
7
10
days in culture
The G-401 cell line has been cloned by limiting dilutions and the cells with partial trisomy
7p were separated from the cells having normal karyotype.
30 passages after subcloning all clones kept their cytogenetic features
The clones with the partial trisomy 7p (1E8 and 3D5) grew more rapidly than the clones
with normal chromosomes (1B11 and E8).
Nubia cell line
Cytogenetic analysis
Cytogenetic analysis of SK-NEP-1 cell line (1)
Cytogenetic analysis of SK-NEP-1 cell line (2)
Cytogenetic analysis of the WT-3a/b cell line (1)
Cytogenetic analysis of the WT 3a/b cell line (2)
Cytogenetic analysis of G-401 cell line (1)
normal Metaphases
Cytogenetic analysis of G-401 cell line (2)
Cells as “Nanofactories”
Organisms of Biotechnological
Interest
Applications: rec.Protein expression
Techniques: Cell Culture
Microorganisms of Biotechnological Interest
Prokaryotes versus Eukaryotes
Prokaryote (bacteria)
• No nuclear membrane
• No membrane bound organelles
• Simple internal structure
Eukaryote (e.g. yeasts, mammals)
• Nuclear membrane
• Numerous membrane bound organelles
• Complex internal structure
Microorganisms of Biotechnological Interest
Fields of Application
Food Biotechnology
Pharmaceutical Biotechnology
Environmental Biotechnology
Microorganisms of Biotechnological Interest
Fields of Application: Environmental Biotechnology
• Microbial Bioremediation
– Cleanup of oil spills with bacteria and
enzymes
• Sewage treatment
– Host of different microorgansims
Microorganisms of Biotechnological Interest
Different Approaches
Use of microorganisms “as they are”
• Brewing / Yogurt etc
Purpose breeding of organisms
4. Production of different compounds e.g. polymers
5. Spillage and waste(water) treatment
•Genetically modified organisms
• Hormones, blood factors: Erythropoietin, Insulin
Applications: Protein expression
Production of (transgenic) proteins
Bacteria (e.g. E. coli)
• Simpler proteins which do not need modifications
such as glycosylations
Yeasts (e.g. S. cerevisiae, P. pastoris)
• Proteins which need modifications
Applications: Protein expression
Production of (transgenic) proteins
Enzymes used in washing powder
• e.g. alpha-amylase, amyloglucosidase,
cellulase, carbohydrase mix, glucose
isomerase, invertase, lactase, pectinase
mix, pectin esterase, chymosin, fungal
‘rennin’, protease, lipase, cellulase
Applications: Protein expression
Production of (transgenic) proteins
Proteins in biomedical research
5. Taq polymerase, restriction
enzymes, DNA ligase and lots of other
DNA modifying enzymes.
6. Antibodies, lysozyme, GFP
Vectors
DNA delivery systems
“Naked” DNA
11. Incorporation
bacterial
of
plasmids
DNA
followed
into
by
transformation
12. Transfection of eukaryotic cells
by lipofection or electroporation
Practical course
Bacterial BioTechniques
Background and aim
13. Introduction of new DNA into
organisms necessary for biotechnological
production and research
14. E. coli easy to handle and
simple
biotechnological
production unit
Practical course
Bacterial Transformation
Vector system
• Use of a plasmid - a circular
extrachromosomal element containing
– origin of replication
– selectable marker (ampicillin resistance)
– gene of interest: His-tagged GFP
Practical course
Bacterial Transformation
Modification and Selection
• Millions of bacteria are use in the
transformation. Only a few will take up the
DNA
•Expressed makers are cotransformed to
identify the few transformants
Practical course
Bacterial Transformation
Make (antibiotic resistance)
or break...
Practical course
Bacterial Transformation
Adsorption of DNA to
bacteria
DNA enters cells
through pores
Recovery and expression of Growth and selection
antibiotic resistance
Maintenance of mammalian cell lines
in vitro
Mammalian Suspension Cells:
Recombinant Cells in Applied Biotechnology
Suspension, Recombinant Cells
• Production of Bio-Pharmaceutics: e.g. recombinant therapeutics
• Diagnostics Industry: e.g recombinant antibodies and recombinant antigens
• Drug Screening: e.g. recombinant receptors & cell-based bioassay
16
“Recombinant” Cells
Definition
Any cell transfected WITH DNA for the expression of a heterologous protein
DNA Transfection in Mammalian Cells;
2 Stable Cell Lines (biopharmaceutical manufacturing)
3 Viral Transfection e.g. baculovirus, adenovirus
• Transient Gene Expression
Fusszeile
04.11.2013
17
Mammalian BioTech Cell Lines ( -S means “suspension adapted” )
CHO-S (Chinese Hamster Ovary cells)
The most widely used mammalian cells for biopharmaceutical
therapeutic manufacturing
HEK 293-S (Human Embryonic Kidney cells)
Most widely used for excellent transient gene expression for
R&D protein production
18
Cell Culture Plate (adherant cells), to Suspension Cell Lines
Adherent Cells in Plate
Suspension Cells PlateSuspension Cells Shacking
19
Cell Count: PCV (Packaged Cell Volume) Tube
The cell density included both viable and nonviable cells.
Factors of conversion from PCV (Packaged
Cell Volume) to cells/ml.
or; “NucleoCounter”
20
“Basic” Culture Medium
RPMI-1640
1.
2.
3.
4.
Inorganic Salts
Amino Acids
Vitamins
Other, glucose,
glutamine as
metabolic
energy source
5. and Fetal Calf
Serum…
21
Cell culture plastic war
Cell culture plastic war
Genes in a Bottle Kit
DNA Extraction Module
Capture Your Essence!
Bottle your DNA! Whether it’s being cloned, sequenced, fingerprinted, mapped, or
genetically engineered, DNA has become an everyday topic in the media and the classroom.
Introduce your students to the molecular framework of biology — with their own DNA!
How do scientists separate pure DNA from cells composed mainly of lipids, proteins,
carbohydrates, and salts? Membranes are first ruptured with detergents to release DNA
into a solution; then proteins and other organic molecules are digested and separated
while retaining intact DNA. The DNA is finally collected by precipitation in a form that can
be manipulated as desired.
With this simple lab activity, students gain practical knowledge by conducting a realworld procedure that is used to extract DNA from many different organisms for a variety
of applications. Your students will extract genomic DNA from their own cheek cells and
watch it precipitate from solution as floating white strands. The DNA strands are then
easily collected and transferred to a glass vial, and the vial is fashioned into a necklace!
Seeing is believing. For students learning about the molecular framework of biology for
the first time, DNA is abstract and intangible. This procedure makes the invisible visible
— seeing their own DNA makes it real and helps students comprehend this previously
invisible substance of life.
Learning opportunities for all levels of instruction. This activity is designed for any
classroom environment and requires no specialized equipment or stains. For secondary
and college level instruction, lessons on DNA structure and function, cell structure, and
enzyme function can be introduced or reinforced with this laboratory activity. For middle
school students, it’s a perfect introduction to the exciting world of DNA science.
We welcome your comments and suggestions. Have fun!
Ingrid Hermanson-Miller, Ph.D.
Biotechnology Explorer
Product Manager
Melissa Woodrow, Ph.D.
Biotechnology Explorer
Scientist
Create context. Reinforce learning. Stay current.
New scientific discoveries and technoligies
create more content for you to teach,
but not more time. Biotechnology
Explorer kits help you teach more
effectively by integrating multiple
core content subjects into a single
lab. Connect concepts with
techniques and put
them into context with
real-world scenarios.
2 Conduct sophisticated
scientific procedures
3 Extract DNA from cheek cells
4 Precipitate and preserve DNA
Environmental
and Health
Science
Scientific
Inquiry
• Genetic testing
• DNA fingerprinting
Genes in
a Bottle
Kit
• Cell structures
• Organelles
• Nuclear and DNA
staining
• Cell organization
• DNA and genetic
variation among
individuals
• Genes are inherited
Structure
and Function
of Organisms
Evolutionary
Biology
Chemistry
of Life
Heredity
and Molecular
Biology
• Chemical properties of
cell components
• Properties of enzymes
• Solubility
• Central dogma:
DNA > RNA > protein > trait
• DNA location, structure, and function
• Basic review of chromosome
inheritance and structure
Table of Contents
Teacher’s Guide
Kit Inventory Checklist ..........................................................................................1
Overview for the Teacher ......................................................................................2
Why Shoud You Teach DNA Extraction? ............................................................2
Intended Audience ............................................................................................2
Curriculum Fit ...................................................................................................3
Recommended Student Background ..................................................................3
Activity Timeline ................................................................................................3
Safety Issues ....................................................................................................3
Keys to Success................................................................................................3
Volume Measurements ......................................................................................3
Background and Fundamentals for Basic Level Instruction ...................................4
Background and Fundamentals for Advanced Level Instruction ............................6
Teacher’s Laboratory Guide
Implementation Timeline ..........................................................................................8
Teacher’s Advance (Pre-Laboratory) Preparation ......................................................8
Workstation Checklist ............................................................................................10
Quick Guide for DNA Extraction and Precipitation ....................................................11
Student Manuals
Basic Level Student Manual ................................................................................13
Introduction .....................................................................................................14
Workstation Checklist ......................................................................................17
Procedure for DNA Extraction and Precipitation .................................................17
Advanced Level Student Manual .........................................................................21
Introduction and Focus Questions ....................................................................22
Workstation Checklist ......................................................................................27
Procedure for DNA Extraction and Precipitation .................................................27
Extension Activities
Dry Laboratory Demonstration of DNA Extraction ..............................................30
Microscropic Observation and Nuclear Staining of Cheek Cells ..........................30
Staining precipitated DNA ................................................................................31
Answers to Focus Questions (Basic Instruction) ...................................................32
Answers to Focus Questions (Advanced Instruction) ............................................33
Teacher’s Guide
Kit Inventory Checklist
This section lists the components provided in this Genes in a Bottle Kit. It also lists
required and optional accessories. Each kit contains sufficient materials to outfit 9
student workstations of up to four students per workstation. Use this checklist to
inventory your supplies before beginning advanced preparation.
Kit Components
Lysis buffer
Powdered protease and salt
15 ml conical tubes
Clear micro test tubes
Multicolor micro test tubes
Disposable plastic transfer pipets
Foam micro test tube holders
Quantity
150 ml
1.5 g
50
60
60
60
10
(✔)
Required Accessories (not included in this kit)
Quantity
(✔)
91% isopropanol (available at drug stores)
or 95% ethanol
Water bath with thermometer, set at 50°C*
Permanent markers
Container of ice
Disposable paper cup or beaker for waste disposal
Beaker or rack to hold 15 m tubes in water bath
(need space for 36 tubes maximum)
approx. 360 ml
❐
1
1–9
1
9
❐
❐
❐
❐
1
❐
❐
❐
❐
❐
❐
❐
❐
Optional DNA Necklace Module** (not included in this kit)
**Each DNA necklace module contains enough material to prepare 18 necklaces. Two
kits are required for a class of 36 students. 166-2200EDU contains:
Glass vials
Silver caps
Plastic plugs
Waxed string
Super glue gel
18
18
18
18
1 tube
* If a temperature-controlled water bath is not available, use one or more insulated
containers (Styrofoam is best) large enough to hold a beaker or rack containing up to
36 15 ml tubes, and fill with water heated to 50°C.
Refills Available Separately
Catalog #
Description
166-2300EDU Genes in a Bottle Kit, contains (1) DNA Extraction Module and (2) DNA
Necklace Modules. Serves up to 36 students
166-2000EDU Genes in Bottle DNA Extraction Module (serves 36 students) 1662200EDU Genes in a Bottle DNA Necklace Module (serves 18 students)
166-2001EDU Genes in a Bottle DNA Extraction Refill Package, includes lysis
buffer and powdered protease + salt
166-2002EDU
Genes in a Bottle Lysis Buffer, 150 ml
1
Cheek Cell DNA Extraction
Capture Your Genetic Essence in a Bottle
Overview for the Teacher
Why Should You Teach DNA Extraction?
1) DNA extraction gives students the opportunity to see their very own
genetic essence.
You and your students will be excited to see the very substance that makes them
unique become visible before their eyes. The precipitated DNA can be sealed and
stored in an attractive glass vial that can be treasured for a long time.
2) DNA extraction helps students to understand properties of DNA.
The DNA molecules that make up our chromosomes are incredibly long and thin.
Ask your students to imagine how such long molecules can fit into microscopic
cheek cells. The fine white fibers that they will see as their DNA precipitates is many
thousands of DNA molecules wound over each other like fibers in yarn.
3) DNA extraction is the first step in DNA technology.
DNA extraction is a routine step in many biotechnology procedures: Gene cloning,
gene mapping, DNA sequencing, and DNA fingerprinting all require that DNA be
extracted and isolated from their cell or tissue sources. With this activity, students can
get an idea of how easily DNA can be isolated for use in cutting-edge research.
Intended Audience
This laboratory is appropriate for students from 5th grade through college, as a first
introduction to DNA or as a quick, easy, and impressive hands-on accompaniment to
existing DNA instruction. Even students who have previously extracted DNA out of
onions or liver will find extracting their own DNA far more relevant and exciting.
The instruction manual includes content for both advanced instruction (9th grade through
college) and basic instruction (5th through 8th grades). Depending on the needs of your
students, you may choose to include activities or background material from either section.
A complete student manual is provided for both levels of instruction.
2
Curriculum Fit
This laboratory activity can be performed at any point during a typical biology or life
science year, but it is particularly relevant when the following topics are being discussed:
•
•
•
•
•
Biomolecules
Cell structure
Mitosis and meiosis
Genetics
DNA technology
Recommended Student Background
High school students should have a general appreciation for the structure and function
of DNA before starting this activity. No prior knowledge of DNA structure or function is
expected for middle school students.
Activity Timeline
This laboratory activity can be performed easily in one 45-minute class period but can
be expanded to include several extension activities.
Lesson 1
Introduction and background material
Lesson 2 Cheek cell isolation, DNA extraction, and precipitation
Lesson 3 DNA necklace preparation (optional)
Safety Issues
In this experiment, no special biosafety handling is required. There is no greater risk of
exposure to infectious agents in this activity than in normal student interactions
(sharing a beverage, sneezing). Students will handle their own biological samples.
Lysis buffer is added to break open the cells, rendering them inviable.
Eating, drinking, smoking, and applying cosmetics are not permitted in the work area.
Wearing protective eyewear and gloves is strongly recommended. Students should
wash their hands with soap before and after this exercise. If any of the solutions gets
into a student’s eyes, flush with water for 15 minutes.
Keys to Success
Ample cell collection is critical for success. For best results, make sure students spend
the recommended amount of time collecting and carefully transferring cheek cells.
Volume Measurements
This kit was developed for use in classrooms with minimal laboratory equipment and
limited knowledge of scientific techniques. Micropipets are not required but can be used
to transfer liquids.
3
Background and Fundamentals for Basic Level
Instruction
What is DNA and what does it do?
Deoxyribonucleic acid (DNA) is a molecule present in all living things, including bacteria,
plants, and animals. DNA carries genetic information that is inherited, or passed down
from parents to offspring. It is sometimes referred to as a biological “blueprint“ because it
determines all of an individual’s physical features such as hair, eye, and skin color,
height, shape of facial features, blood type, and countless others. Your DNA blueprint is
a combination of your mother’s DNA (from her egg) and your father’s DNA (from his
sperm) during conception.
DNA contains four chemical units, referred to by the first letters in their names: A
(adenine), G (guanine), T (thymine), and C (cytosine). These four letters make up a code
for genetic information. The letters of the DNA code function like letters of our alphabet.
The 26 letters in the English alphabet spell words, which can be arranged in infinite ways
to create messages and information. Similarly, the 4 chemical letters of DNA are
organized to make messages that can be understood by cells, called genes. These
genes contain the information to make proteins, which are the basis for almost all of a
body’s and cell’s structures and functions.
Your DNA sequence is the particular arrangement or order of the chemical letters
within your complete DNA collection, or genome. Scientists have determined that
human DNA sequences are 99.9% identical. It is the <0.1% sequence variation from
person to person that makes each of us unique.
Where is DNA found?
With only a few exceptions, DNA is found within practically every cell of an organism’s
body. In our cells, a compartment of the cell called the nucleus contains the DNA. Every
time a cell divides (for growth, repair, or reproduction) the DNA within the cell’s nucleus
is copied and then coiled tightly into chromosomes. The human genetic blueprint is
organized into 46 chromosomes, which contain approximately 40,000 genes that provide
the instructions for constructing the human body.
4
What does DNA look like?
At the molecular level, DNA looks like a twisted ladder or a spiral staircase. The ladder
actually contains two strands of DNA, with pairs of the chemical letters A, G, T, and C
forming the rungs. This structure is called a DNA double helix because of the spiral, or
helical form made by the two DNA strands. Each strand of DNA is very long and thin and
is coiled very tightly to make it fit into the cell’s nucleus. If all 46 chromosomes from a
human cell were uncoiled and placed end to end, the DNA would be 2 meters long —
but only 2 nanometers (2 billionths of a meter) wide.
Fig. 1. A schematic representation of DNA (deoxyribonucleic acid). DNA is a long
chainlike molecule that stores genetic information.
How can we make DNA visible?
We can see our DNA by collecting cells, breaking them open, and condensing the DNA
from all of the cells together. Think of the long, thin DNA molecules as thin white threads.
If the threads were stretched across a room they would be difficult to see, but piled all
together on the floor they would be visible. This laboratory activity uses detergent and
enzymes to break open cells collected from students’ cheeks and release the DNA from
within them. Salt and cold alcohol are then added to make the DNA come out of solution,
or precipitate, into a mass that is big enough to see.
5
Background and Fundamentals for Advanced Level
Instruction
Applications of DNA Technology
This laboratory activity can be integrated into classes that discuss DNA structure and
function and can be used to give students a simple, hands-on experience with their own
DNA. It takes on even more significance if students understand that DNA extraction is
the first step of many biotechnology applications, such as:
Cloning
Cloning means to make many copies of a fragment of DNA or genome. A defective
gene that causes disease may be cloned so that it can be sequenced and analyzed
toward the goal of finding a cure. A gene encoding a desirable protein or trait may be
cloned so that it can be inserted into another organism (see Gene Transfer below).
Likewise, an entire genome can be cloned by inserting it into cell nuclei that are
capable of developing into organisms.
Gene Transfer: Genetically Modified Organisms (GMOs)
To produce useful quantities of a valuable protein, such as a human blood clotting
protein, the gene that codes for the protein is isolated and moved into cells that can be
grown quickly and in quantity. These cell “factories” can be bacteria, yeast, mold, plants,
or animal cells.
Sometimes a mammal is used to produce the desired protein. A gene that codes for a
desirable protein may be inserted into a fertilized cow egg. The genetically modified cow will
produce the desired protein in its milk, from which the desirable protein can be extracted.
Agricultural crops now contain genes from other organisms. For example, some
plants contain a gene that codes for a protein that kills caterpillars. Other plants
contain genes that enable them to withstand herbicides so that farmers can spray a
whole field with herbicide, killing all the weeds and allowing the crop to survive.
DNA Profiling
Using a technique called the polymerase chain reaction (PCR), scientists can study specific
regions of chromosomes where individuals’ DNA sequences differ, and amplify, or make
many copies of them (creating sufficient quantities of these sequences to manipulate and
analyze). Using gel electrophoresis, the differences between individuals can be displayed as
banding patterns that resemble bar codes. This technique can be used to solve crimes, test
paternity, and also to determine the evolutionary relatedness of organisms.
Extraction and Precipitation of DNA: How Does It Work?
Students will start this activity by gently chewing the insides of their cheeks to loosen
cells from the inside of their mouth then rinsing their mouths with water to collect the
cells. Lysis buffer is then added to the solution of cells. The lysis buffer contains a
detergent that breaks apart the phospholipid cell membrane and nuclear membranes,
allowing the DNA to be released. It also contains a buffering agent to maintain the pH of
the solution so that the DNA stays stable.
Protease, an enzyme that digests proteins, is added to remove proteins bound to the
DNA and to destroy cellular enzymes that would digest the DNA. This insures that you
maximize the amount of intact DNA that is extracted. The cell extract containing
protease is incubated at 50°C, the optimum temperature for protease activity.
6
DNA and other cellular components, such as fats, sugars, and proteins, dissolve in the
lysis buffer. DNA has a negative electrical charge due to the phosphate groups on the
DNA backbone, and the electrical charge makes it soluble. When salt is added to the
sample, the positively charged sodium ions of the salt are attracted to the negative
charges of the DNA, neutralizing the electrical charge of the DNA. This allows the DNA
molecules to come together instead of repelling each other. The addition of the cold
alcohol precipitates the DNA since it is insoluble in high salt and alcohol. The DNA
precipitate starts to form visibly as fine white strands at the alcohol layer boundary,
while the other cellular substances remain in solution.
7
Teacher’s Laboratory Guide
This section presents an overview and lesson flow, advance preparation,
student workstation setup, and techniques and concepts to highlight.
Implementation Timeline
1–2 days
Lesson 1
Introduction and background material
Optional dry laboratory demonstration of DNA
extraction — recommended for students in grades 5–8.
See extension activities at the end of the manual.
45 minutes
Lesson 2
30–45 minutes Lesson 3
Cheek cell isolation, DNA extraction, and precipitation
Optional DNA necklace preparation
Teacher’s Advance (Prelaboratory)
Preparation Volume Measurement
This kit contains graduated disposable plastic transfer pipets that will be used for all the
liquid measurements. The diagram below shows marks on the pipet corresponding to the
volumes you will be measuring digital micropipets may also be used.
1 ml
750 µl
500 µl
250 µl
100 µl
2.5 Place the alcohol (isopropanol or ethanol) in the freezer at least 1 hour before
beginning this laboratory.
2.6 Take the pouch containing the powdered protease + salt (‘prot’) and cut open
one corner. Pour the powder into one of the 15 ml tubes. Add 15 ml of water to
the prot. Drinking water works well; distilled water, as used in laboratories, may
be acceptable.
Once the prot is rehydrated, it is good for up to a week if stored in a refrigerator, at
4°C. If you plan to use the kit for several groups of students over a few weeks, it is
recommeded that you measure out some of the protease for use now, and rehydrate
the remaining protease for use later. The protease should be rehydrated at a
concentration of 100 mg/ml.
Aliquot 1.25 ml of the rehydrated prot into 8 pink micro test tubes as described below.
8
Aliquotting of Solutions for Each Student Workstation (4 students/station)
1. For each student, dispense 3 ml of water into a 15 ml tube (up to 4 tubes
per station). Any type of drinking water is acceptable.
2. Dispense 1.25 ml of the rehydrated protease + salt (see p. 8 for dilution
instructions) into 9 pink test tubes and label the tubes “prot”.
3. Dispense 10 ml of lysis buffer into 9 x 15 ml tubes. Label each tube “lysis”.
4. Place 4 x 15 ml tubes of water and one tube of lysis buffer in a cup or test tube
holder, and 1 pink micro test tube labeled “prot” in a foam micro test tube holder at
each student workstation.
Note: Some users may find collecting mouthwash in 15 ml tubes difficult. As an alternative,
instructors may elect to use a small drinking cup to dispense water and collect mouthwash.
9
DNA Extraction and Precipitation
Workstation Checklist
The materials in this kit are sufficient for 36 students.
Teacher’s (Common) Station
Water bath at 50°C with a beaker or rack that can hold up to 36 x 15 ml
tubes Ice-cold bottle of 91% isopropanol or 95% ethanol on ice
Students’ Workstation (4 students per station)
15 ml tubes, each containing 3 ml water
Pink micro test tube labeled “prot”,
containing 1.25 ml of protease + salt
15 ml tube labeled “lysis” containing 10 ml lysis buffer
Disposable plastic transfer pipets
Foam micro test tube holder
Permanent marker
Disposable paper cup or beaker for holding 15 ml tubes
and subsequent waste collection
Number Required
4
1
1
6
1
1
1
Notes to the instructor
Ample cell collection is critical for success. For best results, make sure students spend
the recommended amount of time collecting mouth cells.
10
Quick Guide for DNA Extraction and Precipitation
1. Obtain 15 ml tube containing 3 ml water from
your instructor. Label the tube with your initials.
2. Gently chew the insides of your cheeks for 30
seconds. It is NOT helpful to draw blood!
3. Take the water from the 15 ml tube into your mouth,
and swish the water around vigorously for 30 seconds.
4. Carefully expel the liquid back into the 15 ml tube.
5. Obtain the tube of lysis buffer from your workstation,
and add 2 ml of lysis buffer to your tube.
6. Place the cap on the tube, and gently invert the tube
5 times (don’t shake your tube!). Observe your tube
— do you notice any changes? If you do, write
them down.
7. Obtain the tube of protease (prot) at your
workstation. Add 5 drops of protease to your tube.
11
8. Place the cap on your tube, and gently invert it a
few times.
•
Place your tube in a test tube rack or beaker in the
water bath and incubate at 50°C for 10 minutes.
Remove your tubes from the water bath.
Water bath
50°C for 10 min
•
Obtain the tube of cold alcohol from your instructor
or at the common workstation. Holding your tube at
a 45° angle, fill your tube with cold alcohol, by
adding approximately 10 mls to your tube. It will
take repeated additions to add 10 ml of the cold
alcohol using the disposable plastic transfer pipet.
•
Place your cap on your tube, and let it sit
undisturbed for 5 minutes. Write down anything you
observe happening in the tube.
•
After 5 minutes, slowly invert the tube 5 times to help
the DNA, which has begun to precipitate, to aggegate.
•
With a disposable plastic transfer pipet, carefully
transfer the precipitated DNA along with approximately
750 µl to 1 ml of the alcohol solution into a small glass
vial provided in the DNA necklace kit (166-2200EDU).
If you are not going to make a DNA necklace, save
your DNA in a flip-top tube provided in this kit.
12
Student Manual: Basic Instruction
Cheek Cell DNA Extraction
Capture Your Genetic Essence in a Bottle
Contents
Lesson 1
Introduction and background material, dry laboratory
extension (optional)
Lesson 2
Cheek cell isolation, DNA extraction, and precipitation
Lesson 3
DNA necklace preparation (optional)
GENES IN A BOTTLE: Capture your
unique essence!
Once your students have extracted genomic DNA from
their cheek cells using the DNA Extraction module (1662000-EDU), the DNA strands will be collected and
transferred to a glass vial. The glass vial is then fashioned
into a necklace that can be worn with pride, kept for
posterity, or shared with a loved one! Be the first to wear
DNA on your block! Read more: explorer.bio-rad.com.
The DNA Necklace module contains enough material to
prepare 18 DNA necklaces. Order 2 modules for a class
of 36 students.
Inventory Check List
Amount Provided
Glass vials*
18
Silver caps
18
Plastic stopper caps
18
Waxed cords
18
Super glue gel
1
*vials included in each set may vary
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GENES IN A BOTTLE: Capture your
unique essence!
Instructions
Warning: Since super glue is required for assembling the
DNA necklace, it is suggested that the teacher prepare
the DNA necklaces for younger students. If you
accidentally stick your fingers together, soak the bonded
area with nail polish remover or acetone, then rinse the
area thoroughly. If nail polish remover or acetone is not
available, soak the bonded area in warm soapy water and
gently and slowly roll the skin to break the bond.
•
Using a disposable plastic transfer pipet, carefully
transfer an appropriate portion of the DNA in alcohol
into the glass vial, leaving enough space for the plastic
stopper cap. The glass vial should be filled with alcohol
no higher than ½ cm from the top of the neck of the
vial. Do not fill the entire glass vial with alcohol. (Note
that students can share plastic transfer pipets for
transferring their DNA into the glass vials.)
5
Firmly push the plastic stopper cap into the neck of the
vial to seal the glass vial.
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SDS Gelelectrophoresis
• Electrophoresis : migration of electrically
charged particles in solution or suspension
in the presence of an applied electric field
• Difference in charge and size will lead in
a different electrophoretic mobility
• Electrophoresis can be one- (one property)
or two- (two properties) dimensional
• Support medium is solution or gel-coated plate
2
SDS Gelelectrophoresis
6 Gelelectrophoresis : gel is a crosslinked
polymer whose composition and porosity is
chosen based on the specific weight and
composition of the target
7 Agarose  large nucleic acids
8 Polyacrylamide  proteins or small
nucleic acids
9 SDS-PAGE = Sodium Dodecyl SulfatePolyacrylamide Gelelectrophoresis
3
SDS-Gelelectrophoresis
SDS :
• SDS is the abbreviation for Sodium
Dodecyl Sulfate
• Anionic
detergent
which
denatures
secondary and tertiary structures of proteins
• Gives a negative charge to each protein
in proportion to its mass
4
SDS-Gelelectrophoresis
SDS :
* SDS binds in a ratio of approximately 1.4
g SDS per 1.0 g protein
* Distance of migration through the gel can
be directly related to the size of the protein
5
SDS-Gelelectrophoresis
Acrylamide Polymerization :
4)Polyacrylamide gels are formed by
copolymerization of Acrylamide and
Bis-acrylamide
5)Bisacrylamide acts as a crosslinker
6
SDS-Gelelectrophoresis
Acrylamide Polymerization :
7
SDS-Gelelectrophoresis
Separation :
• Proteins with higher
molecular weight
move more slowly
through the porous
acrylamide gel than
the proteins with
smaller molecular
weight
8
SDS-Gelelectrophoresis
Separating Gel :
2.7 Lower part of cassette
2.8 Concentration 8-15%
Component
Acrylamide Mix
Function
contains Acrylamide, which polymerises into
long chain polymers and Bisacrylamide which is
a crosslinker for the polymers → gel formation
Ammoniumpersulfate an initiator for gel formation (starter radicals)
TEMED
Tris (pH 8.8)
an initiator for gel formation
used as a buffer because it is an innocuos
substance to most proteins
SDS
dissociating agent
9
SDS-Gelelectrophoresis
Stacking (concentrating) Gel :
5. Upper part of cassette
6. The same components
separating gel
7. Less concentrated (5%)
8. Different pH of 6.8
as
in
the
10
SDS-Gelelectrophoresis
Lämmli dye/Sample buffer:
Component
Tris HCl (pH 6.8)
Glycerol
Bromophenol blue
SDS
β-Mercaptoethanol
or DTT
Function
used as a buffer
increase sample density, facilitation gel loading,
preventing migration out of sample wells
visual aid during loading and tracking, dye
allowing easy monitoring of electrophoretic
progress
dissociating agent to denature native proteins to
individual polypeptides;
gives a negative charge to a protein
a reducing agent used to disrupt disulfide bonds
to ensure the protein is fully denatured before
loading on the gel
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SDS-Gelelectrophoresis
Why 3 different pHs and 2 different gels ?
7. Stacking gel at pH 6.8, buffered by Tris-HCl
8. Separating gel buffered to pH 8.8 by Tris-HCl and
9. Electrode buffer (running buffer) at pH 8.3 called Lämmli-buffer.
pH8.3
pH 6.8
pH 8.8
pH 8.3
Glycine can exist in
three different charge
states (positive,
neutral or negative
depending on the pH).
The different buffers
control the charge
state of the glycine
12
SDS-Gelelectrophoresis
• Negatively-charged Glycine ions in the pH
8.3 of the electrode buffer are forced to
enter the stacking gel, where the pH is 6.8.
In this environment Glycine switches
predominantly to the zwitterionic (neutrally
charged) state. This loss of charge causes
them to move very slowly in the electric field
13
SDS-Gelelectrophoresis
-
• The Cl ions (from Tris-HCl) on the other
hand, move much more quickly in the
electric field and they form an ion front that
migrates ahead of the Glycine
-
• Two fronts : the highly mobile Cl front,
followed by the slower, mostly neutral Glycine
front
14
SDS-Gelelectrophoresis
2 All of the proteins in the gel sample have
an electrophoretic mobility that is
intermediate between the extreme of the
mobility of the glycine and Cl
3 Proteins are concentrated into the narrow
zone between the Cl- and glycine fronts
4 Separating gel : the pH switches to 8.8
5 Glycine molecules are mostly negatively
charged and can migrate much faster than
the proteins. So the Glycine front accelerates
the run of the proteins 15
SDS-Gelelectrophoresis
• Result : the proteins are dumped in a very
narrow band at the interface of the stacking
and separating gels and since the
separating gel has an increased Acrylamide
concentration, which slows the movement of
the proteins according to their size, the
separation begins
• Two advantages :
- Aggregation avoided
- Bands are much more sharp
16