Genetic Engineering

Laboratory manual
 DIRECTORIO

MTRO. EMILIO JOSÉ BAÑOS ARDAVIN

RECTOR

MTRO. FRANCISCO FERNANDO EUGENIO URRUTIA ALBISUA

VICE-RECTOR ACADÉMICO

DR. MARIANO SÁNCHEZ CUEVAS

DIRECTOR DEL DEPARTAMENTO DE
CIENCIAS BIOLÓGICAS

M. en C. MARÍA CRISTINA MIRANDA VERGARA

COORDINADORA
DE LA FACULTAD BIOTECNOAMBIENTAL

DR. ELIE GIRGIS EL KASSIS

AUTOR

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 Reflections over Biotechnology and

Genetic Engineering.

We tend to think of biotechnology as a recent

branch of science. The truth is that man has
practiced biotechnology since the dawn of
civilization in order to improve the quality and yield
of domesticated species. Scientific progress has it
that this is done today mainly through genetic
engineering.
However in our modern society the introduction
of a new technology is not always viewed as
progress. Genetic engineering can have a positive
impact on many domains but at the same time it
can, and does, raise objections and ethical
questions. The possibility that a technology is of
great utility does not mean that we can abuse of it.
The opposite is equally true: the fact that abuse is
a possibility does not mean the technology have to
be discarded. The manipulation of living organisms
through genetic engineering cannot be wholly
rejected a priori because it is regarded as
“unnatural” and potentially harmful by some. At
the same time it cannot be accepted based on
purely utilitarian grounds.
We need today to guarantee the beneficial
application of genetic engineering while at the
same time preventing abusive and harmful uses.
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This can only be achieved when our practice of
genetic engineering and biotechnology in general,
is guided by strong bioethical principles.

INDEX PAGE

GENERAL INTRODUCTION………………………………………………………….. 5

LABORATORY SECURITY PROCEDURES ………………………………………. 6

LABORATORY RULES ………………………………………………………………. 7

EVALUATION CRITERIA ………………………………………………………… 8

LABORATORY OBJECTIVES ………………………………………………………. 9

LAB SESSION 1. Isolation of plasmid DNA from bacteria: Miniprep……..….. 10

LAB SESSION 2. Establishing the restriction profile of a plasmid……..……… 14

LAB SESSION 3. Polymerase Chain Reaction (PCR)………….……..………… 18

LAB SESSION 4. Gene cloning……..………………………………………………. 23

LAB SESSION 5. Transformation of competent bacteria……..……………….. 27

LAB SESSION 6. Protein purification using histidine affinity tagging……… 30

LAB SESSION 7. SDS-PAGE…………………………………………..……..…… 33

LAB SESSION 8. Western Blot…………………..……………………………..….. 38

4
GENERAL INTRODUCTION

Life is the greatest of challenges. On our planet this challenge has been answered by an
amazing variety of species in an equally amazing variety of ways. However behind this very rich
biodiversity of species stands one universal molecule: DNA.

DNA encodes for the genetic information organized in the form of genes. The ensemble of
genes, called the genome, constitutes the blueprint of an organism. The genome determines in
great part the properties and the capabilities of the organism. Genetic engineering is based on the
manipulation of this “master plan of life” in order to: i) obtain insights about the functioning of an
organism and ii) improve the properties of an organism of interest and/or introduce novel desirable
traits.
Traditional breeding was until recently the only method for improving the characteristics of
an organism of interest. Two species, each holding an interesting characteristic, are crossed
together and the descendants that present both characteristics are selected. Such selection over
thousands of years has changed marginally useful wild plants into the specialized crops we see
today.
Genetic engineering is gaining ground as the method of choice for improving the traits of an
organism, particularly for crop plants. Instead of relying on sexual recombination, genetic
engineering preserves the integrity of the parental genotype inserting only a small additional piece
of information in the form of a gene that controls a specific trait. Sophisticated biochemical
procedures are employed to “engineer” the foreign gene enabling the host organism to recognize
the new information and used it at the right time, in the proper cellular location, and to the proper
extent. Genetic engineering thus enables researchers to greatly reduce the time needed to
develop the improved organism containing the desired trait. Additionally, it greatly widens the
choice of traits that can be introduced into a new organism. The gene donor organism does not
need to be able to successfully cross with the gene receiver organism as is the case in traditional
breeding. Genetic engineering effectively removes the species barrier.

The process of improving an organism through genetic engineering includes several steps:

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  Step 1: Target identification. Identifying a gene in a donor organism that will confer the
desired trait to the host organism.

 Step 2: Gene cloning. Isolating the gene (through PCR or restriction digestion) and
inserting it into a plasmid. The cloned DNA is called “recombinant DNA”.

 Step 3: Gene engineering. Adding to the gene other DNA sequences (such as a promoter)
that will determine where, when and how the gene will be expressed in the host organism.

 Step 4: Transformation. Transferring the gene to the host organism. A variety of methods
exist that are dependent on the nature of the host organism.

 Step 5: Characterization. The transformed host organism is checked for the correct
functioning of the inserted gene. A successful process would lead to the host organism
acquiring the new trait conferred by the inserted gene.

Scientists have developed various methods and molecular tools in order to achieve each of
these steps. Several of these basic tools will be presented during the various practice sessions.

LABORATORY SECURITY PROCEDURES

 Locate the nearest safety devices.

These devices are items such as fire extinguishers, eyewash, safety shower, fire blankets,
emergency exit, etc. Learn about their operation.

 In case of accident.
Consider the safety of your colleagues. The lab is a place to work seriously. In case of any
accident that result in personal injury or damage to the lab equipment, notify your teacher
immediately.

 Learn about basic security measures.

Laboratory work requires knowledge of a number of basic safety measures that will be
provided by your teacher as necessary.

 Pay attention to the specific security measures.

The experiments performed in some practices require specific safety information. These
instructions will be given by the teacher and/or mentioned in this manual. You must give
them special attention.

 When in doubt, ask the teacher.

When you have questions or doubts do not hesitate to consult with your teacher.
Remember that you are not allowed to perform any experiment that is not authorized by
your teacher.

 Take care of your eyes.

The eye is a particularly susceptible organ. Permanent damage can result from exposure to
corrosive chemicals, splashing particles and UV radiation. It is mandatory to wear safety
glasses whenever you are in lab where the eyes can be exposed to damage. Do not wear
contact lenses in the laboratory since, in case of an accident, chemicals or their vapors can

6
 pass behind the lenses and cause eye injury. Never look directly at a UV lamp, always wear
a protective mask. UV light can cause serious eye injury and complete loss of vision.

 Laboratory costume.
The use of a lab coat is mandatory. No matter the amount of care taken in the execution of
experiments, chemical splashes are inevitable. The lab coat would be preferably made of
cotton as other tissues might adhere to the skin in case of an accident.

 Wear gloves.
It is advisable to wear gloves, especially when using corrosive or toxic substances, or to
avoid contamination with biological agents.

LABORATORY RULES

 All students must wear a white lab coat, correctly buttoned.

 No smoking, eating, drinking or bringing food to the lab, even outside practice hours.

 There will be 15 minutes of tolerance for the check in time. After that students will be denied
access to the laboratory.

 Place your belongings in the place indicated by your teacher. This is to prevent the piling up
of personal belongings in the work area.

 Maintain order during the practice presentation and respect the class and your peers.

 Permanent work teams will be formed. The absence from some of the practices cannot be
compensated by a presence with another group.

 For each performed practice students must submit one report by team that is delivered
within 6 days.

 Each material that is broken or lost shall be replaced with new material (provided with
purchase bill).

 Before starting each practice carefully read the instructions. If there is a part you don’t
understand ask the teacher.

 Each team will be responsible for cleaning used equipment and work area immediately after
the end of the experiment.

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  When you open any substance container (solid or liquid) put the cover back in place
immediately in order to avoid interchanging covers.

 Do not flush solids in the sink. Do not flush hot liquid agar in the sink as it will solidify upon
cooling and plug the sink.

 Biologically contaminated waste should be thrown exclusively in designated waste bins.

 It is strictly forbidden to take out any instrument or laboratory equipment.

 At the end of each practice you should deliver all material or equipment to the teacher.

 At the end of each practice place the chairs back in place and wash your hands with water
and soap.

EVALUATION CRITERIA

The final grade for each period will be calculated according to the following guideline:

 Attitude: 10 %
 Lab reports: 90 %

On the attitude level, will be evaluated the general behavior of the student such as the respect
he/she shows for fellow students and the teacher, punctuality in showing up for the lab sessions,
the care taken in the use of the laboratory equipment, the respect of the security procedures and
the active participation of the student in the execution of the planned experiments among other
things.
The lab report will be written in a Microsoft word format and be delivered using the institutional
email within 6 days of each lab session (before the start of the next session). The lab report should
include the names of the team members, the title of the practice, an introductory paragraph
describing the objectives of the session, a description of the obtained results, including
photographic evidence, and a conclusion section describing the significance of the obtained
results (if the results are positive) or the reasons of the failure of the experiment (if the results are
negative). In addition the lab report will include answers to the questions accompanying each
practice.

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 LABORATORY OBJECTIVES

In this lab the students will be introduced to some basic tools of molecular biology
that are commonly used to manipulate biological macromolecules such as DNA and
proteins. At the end of the practice sessions the student should be able to describe and
implement the various techniques used to manipulate the genome of a living organism and
to study the function of individual genes. The theoretical knowledge gained from the
course combined with the practical knowledge gained from the lab will allow students to
develop a great understanding of the principles and techniques of genetic engineering.

9
PRACTICE 1

Isolation of
plasmid DNA
from
I-INTRODUCTION

bacteria:
A plasmid is a bacterial extra-chromosomal, often circular, DNA molecule that contains non-
essentialMiniprep.
genes for survival under non selective conditions. The plasmids can be easily isolated
from or introduced into bacterial cells. They can be integrated with any desired gene or DNA
fragment. Thus plasmids are of immense use in genetic engineering. Plasmids used by scientists
have typically 3 important elements:

 An origin of replication.
 A selectable marker gene (e.g. resistance to an antibiotic).
 A cloning site (a place to insert foreign DNA).

A B LacZ
Plasmid
Cloning
AmpR site
pUC19

Bacterial chromosome Ori 10

Figure 1.1: Bacterial plasmids.

A) Structure of bacterial DNA. Most, if not all, of the genetic information lies in the single bacterial
 genetic information may lie in one or more small circular DNA molecules called plasmids. B) The
map of a typical plasmid: pUC19. Ori: Origin of replication, AmpR: ampicillin resistance gene,
LacZ: cloning indicator gene.

The origin of replication is a small sequence of DNA at which replication of a plasmid is

initiated. The replication of a plasmid is a process that takes place in a dividing cell and results in
the production of an exact copy of a plasmid. The 2 copies are then distributed among the 2
daughter cells.
The selectable marker gene is usually, but not necessarily, a gene that confers resistance
to a certain antibiotic. It allows maintaining a selective pressure that keeps the plasmids in the
cells. In fact, a cell that contains a plasmid presents a lower growth rate then a plasmid-free cell.
Without the selective pressure imposed by the presence of an antibiotic in the culture the cells
would be tempted to ‘‘kick out’’ the plasmid in order to achieve a higher growth rate. However in
the presence of an antibiotic they need the plasmid and the expression of the antibiotic resistance
gene carried by it in order to survive.
The cloning site is a short stretch of DNA containing specific sequences for restriction
enzymes. It is the point where foreign DNA is inserted (cloned) in order to be multiplied or studied.

The minipreparation of plasmid DNA, or miniprep, is a small-scale isolation and purification

of plasmid DNA from bacteria. Two main approaches have been used to achieve this isolation.
The traditional technique is often a variation on the original protocol by Birnboim and Doly (1979).
The second uses a special silica matrix to bind DNA and then release it under certain conditions.
The matrix is often included in a spin-column. Many of the biotechnology companies sell kits for
plasmid isolation that use this technology. In this practice we will use the Qiagen plasmid
purification Kit that uses the silica matrix technology.

The Qiagen miniprep procedure is based on alkaline lysis of bacterial cells followed by
adsorption of DNA onto silica in the presence of high salt (for more details consult the Qiaprep ®
Miniprep Handbook at www.qiagen.com). The procedure consists of three basic steps:
 Lysis of plasmid-containing bacteria.
 Selective adsorption of DNA onto the silica membrane.
 Washing and elution of plasmid DNA.

Step 1 Step 2 Step 3 Step 4

Pelleting the cells Resuspension in Cell lysis Pelleting the debris
buffer

11

Step 5 Step 6 Step 7 Step 8

 supernatant onto silica membrane
column
Figure 1.2: Schematic representation of the various steps of a minirprep using the silica
membrane technology.

The first two steps of the protocol consist in pelleting the bacteria cells and resuspending
them in a Resuspension Buffer. This buffer contains mainly Tris, EDTA and RNase A. Generally,
the Resuspension Buffer prepares the cells for the next step which is cell lysis using the alkaline
lysis procedure.

Lysis of the cells is done using the lysis buffer that contains NaOH and SDS (sodium
dodecyl sulfate). The lysis buffer work by dissolving the membrane of the cell in order to release
its content including the plasmid.

However letting the plasmid in contact with the lysis solution for too long may damage the
plasmid. For this reason the solution is rapidly neutralized using the Neutralization Buffer that
protects the plasmid from the harsh condition of the lysis buffer and at the same time provide the
ideal conditions for binding of the plasmid onto the silica membrane column. The neutralization
buffer composition is secret and proprietary but it most likely contains Acetic acid, acetic salt and a
chaotropic salt such as guanidine hydrochloride. Chaotropic salts enable denaturing biological
macromolecules such as DNA and proteins and in this case favor the binding of DNA to the silica
membrane. Lowering the pH achieves 2 goals at the same time: neutralizing the alkaline lysis
solution and activating the silica membrane functional groups, in presence of high salt
concentrations, which will bind the plasmid DNA hence the presence of acetic acid and acetic
salts.
After pelleting the cell debris, the plasmid-containing supernatant is transferred to the Spin
column where plasmid DNA will bind to the silica membrane while the rest of the cellular
components will flow through. The Membrane bound DNA is washed using the washing solution in
order to remove the remaining contaminants. The washing solution contains ethanol among others
that allow the DNA to remain on the silica membrane and avoid accidental elution of the plasmid.

The final step of the miniprep process is eluting the plasmid DNA into a small volume,
typically 50 µl. The elution buffer can be ET buffer containing Tris and EDTA at pH 8, or just pure
water. In both cases the elution is done through raising the pH and reducing salt concentration
which result in the detachment of the plasmid from the silica membrane. The Tris-EDTA buffer is
designed to protect the purified plasmid from degradation through accidental contamination with
DNase enzymes. However, ET buffer can interfere with subsequent experiments such as PCR or
sequencing. In this case elution in pure water is preferred.

II-PRACTICE OBJECTIVES

 Understand and describe the various steps of a miniprep procedure.

 Purify a plasmid from a bacterial culture using the Qiagen miniprep kit.
 Determine the quantity and quality of the purified plasmid using a spectrophotometer.

III-MATERIAL

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  Micropipettes of 1000, 200, 20 and 2 µl.
 Corresponding micropipette tips.
 Qiagen miniprep kit.
 1.5 ml eppendorf tubes.
 Racks for eppendorf tubes.
 Spectrophotometer.
 Cuvettes for spectrophotometer.
 Centrifuge.

IV-PROCEDURE
(Adapted from the Qiaprep miniprep handbook).

A- Miniprep:

1. Transfer 1 ml of bacterial culture to a 1.5ml eppendorf tube.

2. Centrifuge for 10 minutes at 10 000 rpm at room temperature.

3. Remove the supernatant containing the culture medium.

4. Resuspend the cells in 250 µl of buffer P1 (resuspension buffer).

5. Add 250 µl of buffer P2 (lysis buffer). Mix gently by inverting the tube for 6 times.

6. Immediately add 350 µl of buffer N3 (neutralization buffer). Mix gently by inverting the tube
for 6 times (the solution should become cloudy).

7. Centrifuge for 15 minutes at 13 000 rpm at room temperature.

8. Apply the supernatant to the Qiaprep spin column.

9. Centrifuge for 60 s. Discard the flow-through.

10. Wash the column by adding 500 µl of buffer PB.

11. Centrifuge for 60 s. Discard the flow-through.

12. Wash the column by adding 750 µl of buffer PE.

13. Centrifuge for 60 s. Discard the flow-through.

14. Centrifuge for an additional 1 min to discard the residual wash buffer.

15. Place the Qiaprep spin column a 1.5 ml eppendorf tube.

16. Add 50 µl of water directly on the silica membrane WITHOUT touching the membrane.

17. Let the column stand for 1 min.

18. Centrifuge for 60 s. Discard the column.

B- Determining plasmid quantity:

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 19. In a new 1.5 ml eppendorf tube mix: 2 µl of plasmid solution, 198 µl of water.

20. Also prepare a blank sample containing 200 µl of water.

21. Use the blank sample to set the baseline in the spectrophotometer.

22. Measure the absorbance of the diluted plasmid solution at 260 nm.

23. Use the following formula to calculate the amount of plasmid:

[DNA] in µg/ml = OD260nm x dilution factor x 50

V-Questions:
1. Report the concentration of the plasmid you isolated.
2. Describe the function of each of the components of the Resuspension Buffer.
3. Research and report the difference between a miniprep, a midiprep and a maxiprep.
4. If the purpose of your miniprep experiment is long term storage of the plasmid in the freezer
which solution would you use for the final elution step: water or TE buffer? Argument your
choice.
5. Research and report the mechanism of action of ampicillin resistance.

REFERENCES
Birnboim, HC and Doly J (1979) A rapid alkaline procedure for screening recombinant plasmid DNA. Nucleic Acids
Res. 7: 1513-1523.
PRACTICE 2

Establishing
the
restriction
I-INTRODUCTION

profile
Restriction of aare DNA-cutting enzymes found in bacteria. Because they cut within
enzymes
plasmid.
the molecule, they are also called restriction endonucleases. Restriction enzymes are fundamental
tools in genetic engineering. They are an important part of the tools set available to scientists that
allow them to cut (restriction enzymes), paste (DNA ligases) and synthetize (DNA polymerases)
DNA fragments.

A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides
called restriction sites specific for each enzyme. For example, the bacterium Serratia marcescens
produces an enzyme named SmaI that cuts DNA wherever it encounters the sequence:

5’-CCCGGG-3’ SmaI 5’-CCC GGG-3’

3’-GGGCCC-5’ 3’-GGG CCC-5’

The cut is made between the adjacent C and G (the location of the cut is represented by the
red arrow). This type of restriction enzymes produces what are called “blunt-end” fragments. But
often the enzyme does not execute the cut in the middle of the restriction site. For example the
enzyme BamHI recognizes the following restriction site:

5’-GGATCC-3’ BamHI 5’-C CCGGG-3’

3’-CCTAGG-5’ 3’-GGGCC C-5’

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 The cut is made between the adjacent Gs. This type of restriction enzymes produces what
are called “Sticky-end” fragments. The sticky-end fragments produced by the same restriction
enzymes can be joined back together using an enzyme called DNA ligase. Thus restriction
enzymes and ligases are essential tools that allow scientist to cut and paste any DNA fragments.

A plasmid DNA can contain one or more restriction sites for each restriction enzyme. The
ensemble of restriction sites of all the enzymes constitutes a restriction map (figure 2.1).

Figure 2.1: A partial restriction map of

the plasmid pUC19.

The presence and distribution of restriction sites are dependent on the sequence of a
plasmid. Since each plasmid have a unique sequence the nature, number and distribution of
restriction sites are unique to each plasmid. Thus a restriction profile is unique to each plasmid
and can be used both to identify the plasmid (like a fingerprint) and to plan cloning strategies.

Once a plasmid has been cut by a restriction enzyme one or several DNA fragments of
various sizes are produced. However these fragments remain mixed in solution and need to be
separated in order to determine their size. Agarose gel electrophoresis is the most wildly used
technique to separate DNA fragments.

When an agarose gel is made, agarose forms a three-dimensional net with pores more or
less the same size, the latter being a function of the agarose concentration. When an electric
current is applied to the gel, negatively-charged DNA fragments deposited in a well travel through
the gel towards the positively charged cathode. DNA fragments will travel more or less easily
through the gel pores depending on their size, small fragments travelling faster and farther then
big fragments. Thus DNA fragments are separated based on their size. The size of the fragments
is then determined by comparison to a standard containing DNA fragments of known sizes.

When an uncut plasmid is deposited on an agarose gel we might expect to observe one
band of DNA, but in reality we can observe up to three bands.

II-PRACTICE OBJECTIVES

 Understand the importance and uses of restriction enzymes.

 Perform a digestion of a plasmid with several restriction enzymes.
 Use agarose electrophoresis in order to separate DNA fragments and determine their size.
 Establish a restriction map of the plasmid.

15
III-MATERIAL

 Micropipettes of 1000, 200, 20 and 2 µl.

 Corresponding micropipette tips.
 Water bath.
 1.5 ml eppendorf tubes.
 Racks for eppendorf tubes.
 Plasmid.
 Restriction enzymes.
 10x loading dye.
 Agarose.
 TAE buffer.
 Spatula.
 Balance.
 Electrophoresis equipment.
 Microwave oven.

IV-PROCEDURE

A- Digestion of the plasmid by restriction enzymes:

1. In a 1.5 ml eppendorf tube mix:

Plasmid : 5 µl.
Enzyme1 : 1 µl.
Enzyme2 : 1 µl.
Buffer : 2 µl.
Water : 11 µl.

2. Also prepare a control undigested plasmid sample containing:

Plasmid : 5 µl.
Buffer : 2 µl.
Water : 13 µl.

3. Mix by pipetting.

4. Incubate the tubes in a water bath set to 37°C for 15 minutes.

B- Agarose gel electrophoresis:

5. You need to prepare 1 % agarose gel i.e. 1 gram of agarose in 100 ml of TAE buffer. For
that purpose weigh 1 gram of agarose and then put them in 100 ml of TAE.

6. Heat the agarose solution in a microwave oven for 4 minutes. If the agarose is not
completely dissolved shake the bottle and heat for one additional minute.

7. Let the bottle cool down until you can hold it with your hands for more than 10 sec without
burning your hands. That means the temperature of the agarose is around 50 to 55°C.

8. Prepare the agarose mold by adjusting the well comb and verifying the mold is horizontal.

9. Pour the molten agarose into the mold until the thickness of the gel is around 4 mm.

10. Wait 20 minutes for the gel to solidify. And then carefully remove the mold.

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 11. Put the gel in the electrophoresis apparatus then fill it with TAE buffer until the thickness of
the solution above the gel equals the thickness of the gel.

12. Let the gel incubate in TAE for 5 minutes before loading the samples.

13. To each 20 µl of digestion sample add 2 µl of 10x loading dye and mix by pipetting.

14. In the first well load 5 µl of the DNA marker.

15. In subsequent wells load 15 µl of each sample in a different well.

16. Put the lid of the electrophoresis cuvette respecting the colors the cathodes and anodes.

17. Apply a current of 120 volts for 45 minutes.

18. Turn off the electric generator and remove the lid of the cuvette.

19. Put on nitrile gloves.

20. Remove the gel carefully and put it in the ethidium bromide solution. Turn the shaker on
and let incubate for 10 minutes.

21. Transfer the gel to a water solution and let agitate in water for another 10 minutes.

22. Put the gel in the transilluminator.

23. Close the door of the transilluminator to protect yourself from UV light.

24. Turn UV on.

25. Observe the results and take an image of the gel and save it in a file.

V-Questions:

1. Present the results of the gel electrophoresis.

2. Determine the relative position of the restriction sites on an approximate plasmid map.
3. What is the reason for observing several bands in the uncut plasmid lane?
4. During gel electrophoresis bubbles are observed at the cathode and anode. What are the
gases that form them and how that explains that there is more bubbles at the anode then
the cathode’s side?
5. Describe the mechanism by which ethidium bromide can make DNA visible under UV light.

17
PRACTICE 3

Polymerase
I-INTRODUCTION Chain
Reaction
Sometimes called "molecular photocopying," the polymerase chain reaction (PCR) is a fast
(PCR).
and inexpensive technique used to "amplify", or make many copies of, small segments of DNA.
Because significant amounts of a sample of DNA are necessary for molecular and genetic
analyses, studies of isolated pieces of DNA are nearly impossible without PCR amplification. Once
amplified, PCR products can be used in many different laboratory procedures. A particularly useful
feature of PCR is that it allows the amplification process to be limited to specifically targeted
segments of the DNA mixture. PCR is generally considered one of the most important scientific
advances in molecular biology. PCR revolutionized the study of DNA to such an extent that its
creator, Kary Mullis, was awarded the Nobel Prize for Chemistry in 1993.

PCR is a process based on the ability of a DNA polymerase enzyme that can synthesize a
complementary strand to a targeted segment of DNA in a test tube mixture of the four DNA bases.
In addition, the mixture must also contain two DNA fragments, each about 20 bases long, called
primers, which have sequences complementary to areas adjacent to each side of the target
sequence. (To do PCR, you need to know the DNA sequence around the region you want to
amplify). These primers can be constructed in the lab but are generally purchased from
commercial suppliers. If chosen well, the 20-25 base pair sequence will be unique and will match
only the place specifically chosen thus limiting and defining the area to be copied.

PCR is closely patterned after the natural principle of DNA replication. It is a three-step
process, referred to as a cycle, that is repeated a specified number of times. One PCR cycle
consists of the following steps:
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  Denaturation: During this initial step, heat (usually hotter than 90°C) separates
double-stranded DNA into two single strands. This process is called "denaturation."
Denaturation is possible because the hydrogen bonds linking the bases to one
another are weak. The hydrogen bonds break at high temperatures, whereas the
bonds between deoxyribose and phosphates, which are stronger covalent bonds,
remain intact.
 Annealing: The goal of PCR is not to replicate the entire strand of DNA but to
replicate a target sequence (the size of which can vary from 100 bp to 25000 bp)
unique to the organism being studied. Targeting the sequence is achieved by using
primers. Primers mark the ends of the target sequence. Two primers are included in
the PCR, one for each of the complementary single DNA strands that was produced
during denaturation. The beginning of the DNA target sequence of interest is marked
by the primers that anneal (bind) to the complementary sequence. With regard to
temperature, annealing usually takes place between 40 °C and 65 °C, depending on
the length and base sequence of the primers. This allows the primers to anneal to
the target sequence with high specificity.
 Extension: Once the primers anneal to the complementary DNA sequences, the
temperature is raised to approximately 72 °C and a DNA polymerase, ex: Taq DNA
polymerase, is used to replicate the DNA strands. Taq DNA polymerase, begins the
synthesis process at the region marked by the primers. It synthesizes new double
stranded DNA molecules, both identical to the original double stranded target DNA
region, by facilitating the binding and joining of the complementary nucleotides that
are free in solution (dNTPs). Extension always begins at the 3' end of the primer
making a double strand out of each of the two single strands. Taq DNA polymerase
synthesizes exclusively in the 5' to 3' direction. Therefore, free nucleotides in the
solution are only added to the 3' end of the primers constructing the complementary
strand of the targeted DNA sequence.

Repeated heating and cooling cycles multiply the target DNA exponentially, since each new
double strand separates to become two templates for further synthesis. In about 1 hour, 20 PCR
cycles can amplify the target by a million fold. In 32 cycles at 100% efficiency, 1.07 billion copies
of targeted DNA region are created.

The entire cycling process of PCR is automated and can be completed in just a few hours.
It is directed by a machine called a thermocycler, which is programmed to alter the temperature of
the reaction as necessary to allow DNA denaturing and synthesis. The instruments vary in the
number of samples that can be handled, the size of reaction tube used, the speed of temperature
change, and the cost.

19
 Figure 3.1: The 3 steps of PCR: denaturation, primer annealing, and extension. The
temperature of primer annealing step is an example and vary with each primer couple.

II-PRACTICE OBJECTIVES

 Understand the principle and uses of PCR.

 Understand the properties of DNA polymerases.
 Amplify the mouse dihydrofolate reductase (DHFR) gene.
 Use agarose gel electrophoresis to visualize the results of the PCR.
 Determine the size and integrity of the amplified gene.

III-MATERIAL

 Micropipettes of 1000, 200, 20 and 2 µl.

 Corresponding micropipette tips.
 0.2 ml PCR tubes.
 1.5 ml eppendorf tubes.
 Racks for eppendorf tubes.
20
  Plasmid.
 Primers
 Phusion polymerase kit.
 10x loading dye.
 Agarose.
 TAE buffer.
 Spatula.
 Balance.
 Electrophoresis equipment.
 Microwave oven.

IV-PROCEDURE

A- Running the PCR:

1. Let tubes containing the necessary PCR ingredients thaw at room temperature and then
place them on ice (except the tube containing the Phusion polymerase that you should
NEVER let at room temperature).

2. Vortex all tubes except water and the tube containing the Phusion polymerase.

3. In a 0.2 ml PCR tube mix:

Plasmid 0.4 µl
5x Phusion HF buffer 4 µl
Primers 10 µM 1 µl
dNTP 10 mM 0.4 µl
Phusion polymerase 0.2 µl
Water 14 µl

4. Maintain the PCR tubes on ice.

5. Turn on the PCR machine.

6. Load and run the following program:

Step 1: heat the block until 98°C. Prepare the block to receive the
PCR tubes.
Step 2: Hold at 98°C until further notice.

Step 3: maintain at 98°C for 30 sec. Initial denaturation.

Step 4: maintain at 98°C for 10 sec. Denaturation step

Step 5: maintain at X°C for 15 sec. Annealing step Cycle steps.

Step 6: maintain at 72°C for 10 sec. Elongation step

Step 7: repeat 29 times steps 4, 5 and 6. Number of cycles.

Final extension. 21

End of PCR.
 Step 8: maintain at 72°C for 5 min.

Step 9: Hold at 4°C until further notice.

7. Let the PCR lid heat to 105°C to avoid evaporation of the PCR solution.

8. Put the PCR tubes in the block and close the lid.

9. Instruct the machine to pursue the program that has paused at step 2.

B- Perform the agarose gel electrophoresis:

10. Weigh 1 gram of agarose and then put them in 100 ml of TAE.

11. Heat the agarose solution in a microwave oven for 4 minutes. If the agarose is not
completely dissolved shake the bottle and heat for one additional minute.

12. Let the bottle cool down until you can hold it with your hands for more than 10 sec without
burning your hands.

13. Prepare the agarose mold by adjusting the well comb and verifying the mold is horizontal.

14. Pour the molten agarose into the mold until the thickness of the gel is around 4 mm.

15. Wait 20 minutes for the gel to solidify. And then carefully remove the mold.

16. Put the gel in the electrophoresis apparatus then fill it with TAE buffer until the thickness of
the solution above the gel equals the thickness of the gel.

17. Let the gel incubate in TAE for 5 minutes before loading the samples.

18. To each 20 µl of PCR sample add 2 µl of 10x loading dye and mix by pipetting.

19. In the first well load 5 µl of the DNA marker.

20. In subsequent wells load 15 µl of each sample in a different well.

21. Put the lid of the electrophoresis cuvette respecting the colors the cathodes and anodes.

22. Apply a current of 120 volts for 45 minutes.

23. Turn off the electric generator and remove the lid of the cuvette.

24. Put on nitrile gloves.

25. Remove the gel carefully and put it in the ethidium bromide solution. Turn the shaker on
and let incubate for 10 minutes.

26. Transfer the gel to a water solution and let agitate in water for another 10 minutes.

27. Put the gel in the transilluminator.

22
 28. Close the door of the transilluminator to protect yourself from UV light.

29. Turn UV on.

30. Observe the results and take an image of the gel and save it in a file.

V-Questions:

1. Present the results of the gel electrophoresis. Does the amplified DNA fragment present the
expected size? Based on your answer do you estimate that the amplified fragment
represent the target gene?
2. Describe the mechanism that allows a thermocycler to change temperatures rapidly.
3. What is the function of the heated lit?
4. Proteins are usually denatured at temperatures higher than 60°C. Why the DNA
polymerase is not denatured by the high temperatures used during a typical PCR cycle.

PRACTICE 4

Gene
I-INTRODUCTION cloning.
Using modern techniques in biotechnology, genetic engineers can alter an organism’s traits
(causing bacteria to produce insulin for example or plants to produce their own insecticides) by
directly manipulating the organism’s genes. This process often involves transferring a particular
gene form one organism to another. This requires that the gene to be transferred be isolated and
copied to produce sufficient numbers of the gene. Genetic engineers use the process of gene
cloning to isolate and copy specific genes within an organism’s DNA.

In order to be cloned, a gene needs to be inserted into a plasmid. The plasmid plays a dual
role: making many copies of the gene and serving as a transfer vector from the DNA of origin to
23
the target DNA hence the plasmid’s alternative name as vector. But prior to the cloning step the
gene needs to be isolated from the DNA of origin this is usually done using either restriction
enzymes or Polymerase Chain Reaction (PCR).

Traditionally the cloning of the DNA fragment in a plasmid is done be cutting them both with
the same restriction enzyme or enzymes and then ligating together the resulting fragments using
the enzyme DNA ligase. However the method that we will use is a novel non-traditional method
that relies on homologous recombination in order to insert the desired DNA fragment in a plasmid
using the In-Fusion cloning kits. These kits allow the cloning of any PCR fragment into any
linearized vector in a single step without restriction digestion of the PCR fragment or ligation. The
ability to clone directly into any vector at any restriction site eliminates the need for any further
subcloning or manipulation. With the In-Fusion system, it is possible to create modular expression
vectors with interchangeable parts, construct fusion proteins, delete and replace DNA segments,
insert point mutations, make internal fluorescent protein fusions, swap tags on a gene, add UTRs
to a cDNA, etc.

Figure 4.1: Cloning strategy using In-fusion technology.

The method relies on the In-Fusion enzyme, a protein that can fuse PCR-generated
sequences to linearized vectors efficiently and precisely by recognizing a 15 bp overlap at their
ends. First, a PCR product containing the DNA fragement of interest is generated using a high-
fidelity DNA polymerase. The primers that are used for this amplification step must have at least
15 bp of their 5’ (for the forward primer) and 3’ (for the reverse primer) ends that are identical to
the extremities of the digested cloning plasmid. The PCR product is then simply combined to the
linearized vector with the In-Fusion enzyme and incubated at room temperature before bacteria
transformation.

24
 Figure 4.2: Cloning multiple DNA fragments in a single step using In-Fusion technology.

II-PRACTICE OBJECTIVES

 Understand the role of the various enzymes used in gene cloning.

 Design a cloning strategy using the In-Fusion technology.
 Clone the mouse dihydrofolate reductase (DHFR) gene in the pGEM-T vector using the In-
Fusion technology.

III-MATERIAL

 Micropipettes of 1000, 200, 20 and 2 µl.

 Corresponding micropipette tips.
 1.5 ml eppendorf tubes.
 Racks for eppendorf tubes.
 Plasmid.
 Infusion cloning kit.
 Block Heater.
 Water bath.
 100 ml glass beaker.
 Millipore filter.

IV-PROCEDURE

A- Digestion of the pGEM-3Z plasmid with XbaI and HindIII:

1. In a 1.5 ml eppendorf tube mix:

Plasmid: 15 µl.
XbaI: 1 µl.
HindIII: 1 µl.
Buffer E: 3 µl.
Water: 10 µl.

2. Mix by pipetting.

3. Incubate the tubes in a water bath set to 37°C for 20 minutes.

4. Inactivate the restriction enzymes by incubating the digestion solution at 65°C for 15
minutes.

B- Amplifying the DFHR gene with In-fusion specific primers:

5. Let tubes containing the necessary PCR ingredients thaw at room temperature and then
place them on ice (except the tube containing the Phusion polymerase that you should
NEVER let at room temperature).

25
6. Vortex all tubes except water and the tube containing the Phusion polymerase.

7. In a 0.2 ml PCR tube mix:

Plasmid with DHFR gene 0.4 µl
5x Phusion HF buffer 4 µl
Primers 10 µM 1 µl
dNTP 10 mM 0.4 µl
Phusion polymerase 0.2 µl
Water 14 µl

8. Maintain the PCR tubes on ice.

9. Turn on the PCR machine.

10. Load and run the following program:

Step 1: heat the block until 98°C.

Step 2: Hold at 98°C until further notice.

Step 3: maintain at 98°C for 30 sec.

Step 4: maintain at 98°C for 10 sec.

Step 5: maintain at X°C for 15 sec.

Step 6: maintain at 72°C for 10 sec.

Step 7: repeat 29 times steps 4, 5 and 6.

Step 8: maintain at 72°C for 5 min.

Step 9: Hold at 4°C until further notice.

11. Let the PCR lid heat to 105°C to avoid evaporation of the PCR solution.

12. Put the PCR tubes in the block and close the lid.

13. Instruct the machine to pursue the program that has paused at step 2.

C- Purifying the PCR and restriction digestion products:

14. Pour pure water in 100 ml glass beaker.

15. Place a Millipore filter on the surface of the water without submerging it.

16. Use a pipette to place the solution containing the PCR product on the center of the filter and
the plasmid solution on the side of the filter.

17. Let the drops stand on the filter for 10 minutes.

18. Using a pipette transfer the purified PCR and digestion solutions to new 1.5 ml eppendorf
tubes.

26
 D- Cloning the DHFR gene into pGEM-3Z using the In-Fusion technology:

19. Set up an In-Fusiom cloning reaction by adding the following to one In-Fusion pellet:
PCR product: 4 µl.
Digested plasmid: 4 µl.
Water: 2 µl.

20. Incubate the reaction for 15 minutes at 37°C then for 15 minutes at 50°C.

21. Place the reaction at -20°C.

V-Questions:

1. What are the advantages and disadvantages of both the traditional cloning method and the
in-Fusion based cloning method?
2. Why it is better to use 2 restriction enzymes for cloning instead of just one?
3. Describe the principle behind the purification of DNA using Millipore filters?

PRACTICE 5

Transformati
on of
I-INTRODUCTION competent

bacteria.
Genetic engineering relies on a many microbiological procedures such the culture and
transformation of competent bacteria. A professional Genetic Engineer must master many aspects
of microbiology including working properly in a sterile environment.

Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a
bacterium. Bacterial cells into which foreign DNA can be transformed are called competent. Some
bacteria are naturally competent (e.g Bacillus subtilis), whereas others (such as Escherichia coli)

27
are not naturally competent. Non-competent cells can be made competent and then transformed
via one of two main approaches; chemical transformation and electroporation.

Figure 5.1: Principle of

preparation and heat
shock transformation of
chemically competent
bacteria.

Figure 5.2: Transformed bacteria culture

on solid media.

II-PRACTICE OBJECTIVES

 Understand the role of competent bacteria in the cloning process.

 Describe the process by which bacteria can be made competent for transformation.
 Transform the chemically competent bacterial strain JM109 with the cloning product from
the previous practice.
 Select the transformed bacteria using an appropriate growth medium.
 Acquire good practices for work under sterile conditions.

III-MATERIAL

 Micropipettes of 1000, 200, 20 and 2 µl.

 Corresponding micropipette tips.
 Sterile LB medium.
28
  Sterile LB Agar medium containing Ampicillin.
 Rack for eppendorf tubes.
 Cloning product (practice 4).
 JM109 competent bacteria.
 Water bath.
 Ice bucket.
 Horizontal laminar flux sterile hood.
 Glass Pasteur pipettes.
 Sterile 15 ml tubes.
 Rack for 15 ml tubes.
 Agitator/Incubator for bacteria.

IV-PROCEDURE

A- Thermal shock transformation of competent bacteria:

1. Chill sterile 15 ml tubes on ice.

2. Remove frozen competent cells from -70°C and place on ice until just thawed.

3. Transfer 100 µl of competent cells to the chilled 15 ml tube.

4. Add 2.5 µl of the cloning product to the competent cells.

5. Return the tubes on ice for 10 minutes.

6. Heat shock the cells for 45 seconds in a water bath at exactly 42°C.

7. Return the tubes on ice for 2 minutes without shaking.

8. Add 900 µl of cold (4°C) sterile LB medium.

9. Incubate for 1 hour at 37°C with shaking.

B- Growth of transformed bacteria on selective medium:

10. Incubate the petri dishes containing LB agar medium supplemented with ampicillin at 37°C
for at least 30 minutes.

11. Deposit 300 µl of the bacteria culture on the petri dish containing the LB agar.

12. Gently spread the bacteria on the surface of the culture medium using sterile Pasteur
pipette. Be careful no to damage the growth medium.

13. Put the lid back on the petri dishes and close it with parafilm to maintain sterility.

14. Incubate the petri dishes overnight at 37°C without shaking.

V-Questions:

1. Present the results of the transformation experiment with photos?

2. What is the principle of electroporation?

29
 3. What are the advantages and disadvantages of chemical transformation and
electroporation?
4. What is the principle behind blue/white cloning?

PRACTICE 6

Protein
purification
using
I-INTRODUCTION

histidine
The DNA sequence specifying a string of six to nine histidine residues is frequently used in
vectors foraffinity
production of recombinant proteins. The result is expression of a recombinant protein
with a 6xHis or poly-His tag fused to its N- or C-terminus.
tagging.
30
 Expressed His-tagged proteins can be purified and detected easily because the string of
histidine residues binds to several types of immobilized metal ions, including nickel, cobalt and
copper, under specific buffer conditions. In addition, anti-His-tag antibodies are commercially
available for use in assay methods involving His-tagged proteins. In either case, the tag provides a
means of specifically purifying or detecting the recombinant protein without a protein-specific
antibody or probe.

Figure 6.2: Typical expression vectors with or

Histidine tag fusion at the N-terminal or C-
terminal side of the protein.

Figure 6.1: Principle of affinity based chromatography.

II-PRACTICE OBJECTIVES

 Describe the use of affinity tagging to purify a specific protein.

 Differentiate native and denaturing purification conditions.
 Extract and purify the mouse dihydrofolate reducatse (DHFR) tagged with a 6xHis tag using
the Ni-NTA protein purification kit from Qiagen.
 Quantify the purified protein using the Bradford reagent.

III-MATERIAL

 Micropipettes of 1000, 200, 20 and 2 µl.

 Corresponding micropipette tips.
 Rack for eppendorf tubes.
 1.5 ml eppendorf tubes.
 Buffer B.
 Buffer C.
 Buffer E.
 Refrigerated centrifuge.
 Agitator.
 Spectrophotometer.

IV-PROCEDURE

A- Protein purification from frozen E. coli cells under denaturing conditions:

31
 1. Thaw cells for 15 min and Resuspend in 700 µl of buffer B.

2. Incubate cells with agitation for 15 minutes at room temperature. When lysis is complete the
solution should become translucent.

3. Centrifuge lysate at 12000 g for 20 minutes at room temperature (20-25°C) to pellet the cell
debris. Collect the supernatant. Save 20 µl of the cleared lysate for SDS-PAGE analysis.

4. Equilibrate a Ni-NTA spin column with 600 µl buffer B.

5. Centrifuge for 2 min at 890 g.

6. Load up to 600 µl of the cleared lysate supernatant containing the 6xHis-tagged protein
onto a pre-equilibrated Ni-NTA spin column.

7. Centrifuge for 5 min at 270 g and collect the flow-through. Save the flow-through for
analysis by SDS-PAGE to check binding.

8. Wash the NiNTA spin column with 600 µl Buffer C.

9. Centrifuge for 2 min at 890 g.

10. Wash the Ni-NTA spin column with 600 µl a second time.

11. Centrifuge for 2 min at 890 g.

12. Elute the protein twice with 200 µl buffer E. Centrifuge for 2 min at 890 g and collect the
eluate.

B- Estimation of the quantity of purified protein using the Bradford reagent:

13. Mix the following in a cuvette:

Bradford reagent 200 µl

Proteins extract or water 10 µl
Water 790 µl
Total 1000 µl

14. Let the color develop at room temperature for 5 minutes.

15. Read the absorbance at 595 nm.

16. Use the BSA standard curve that will be provided to you in order to estimate protein
concentration.

Questions:

1. Present the results including the quantity of purified protein and photos of the Bradford
reaction.
2. What metal does the protein purification kit used in this experiment employs?
3. Is it possible to purify several proteins using the same column? Argument your answer.
4. What other tags can be used to purify proteins?

32
PRACTICE 7

SDS-PAGE.

I-INTRODUCTION

The purpose of SDS-PAGE is to separate proteins according to their size, and no other physical
feature. In order to understand how this works, we have to understand the two halves of the name:
SDS and PAGE.

33
SDS
Since we are trying to separate many different protein molecules of different shapes and sizes, we
first want to denature them so that the proteins no longer have any secondary, tertiary or
quaternary structure (i.e. we want them to retain only their primary amino acid structure). Consider
two proteins that are each 500 amino acids long but one is shaped like a closed umbrella while the
other one looks like an open umbrella. If you try to run down the street with both of these
molecules under your arms, which one would be more likely to slow you down, even though they
weigh exactly the same? This analogy illustrates how mass and the 3D structure of a molecule will
determine how well it can move through an environment. We use SDS to denature all proteins to
the same linear shape.
SDS (sodium dodecyl sulfate) is a detergent (soap) that can dissolve hydrophobic molecules but
also has a negative charge attached to it. Therefore, if a cell is incubated with SDS, the
membranes will be dissolved, all the proteins will be solubilized by the detergent, plus all the
proteins will be covered with many negative charges. So a protein that started out like the one
shown in the top part of figure 7.1 will be converted into the one shown in the bottom part of figure
7.1. The end result has two important features: 1) all proteins retain only their primary structure
and 2) all proteins have a large negative charge which means they will all migrate towards the
positive pole when placed in an electric field.

Figure 7.1: SDS effect on proteins. SDS denature the 3D structure of proteins and give them a
uniform negative charge.

34

Figure 7.2: principle of polyacrylamide gel electrophoresis (PAGE).

PAGE
If the proteins are denatured and put into an electric field, they will all move towards the positive
pole at the same rate, with no separation by size. So we need to put the proteins into an
environment that will allow different sized proteins to move at different rates. The environment of
choice is polyacrylamide, which is a polymer of acrylamide monomers. When this polymer is
formed, it turns into a gel and we will use electricity to pull the proteins through the gel so the
entire process is called polyacrylamide gel electrophoresis (PAGE). A polyacrylamide gel is not
solid but is made of a labyrinth of tunnels through a meshwork of fibers (figure 7.2).
We apply the mixture of denatured proteins to the gel and apply the current. If all the proteins
enter the gel at the same time and have the same force pulling them towards the other end, which
ones will be able to move through the gel faster? Think of the gel as a tiny forest with many
branches and twigs throughout the forest but they form tunnels of different sizes. If we let children
and adults run through this forest at the same time, who will be able to get through faster? The
children of course. Why? Because of their small size, they move through the forest faster since
they have access to more of the paths in the forest while adults are limited to only the larger paths.
Likewise, small molecules can maneuver through the polyacrylamide forest faster than big
molecules.

You have to remember that when we work with proteins, we work with many copies of each type
of protein. As a result, the collection of proteins of any given size tends to move through the gel at
the same rate, even if they do not take exactly the same tunnels to get through. Back to our
analogy of the forest. If we were in a hot air balloon above the forest and watched 100 children,
100 teenagers, and 100 large adults running through the forest, we would see a collection (or
band) of children moving quickly though each individual would choose his or her own route
through the forest. Behind the smallest individuals, we would see a band of teenagers moving
slower, and a third band made of adults plodding their way through the forest using only the
largest of paths. Likewise, proteins tend to move through a gel in bunches, or bands, since there
are so many copies of each protein and they are all the same shape and size. When running an
SDS-PAGE, we never let the proteins electrophorese “run” so long that they actually reach the
other side of the gel. We turn off the current and then stain the proteins and see how far they
moved through the gel (until we stain them, they are colorless and thus invisible). Figure 7.3
shows a cartoon gel. Notice that the actual bands are equal in size, but the proteins within each
band are of different sizes.

35
 Figure 7.3: Top view of a theoretical SDS PAGE after the current has been on for a while (positive
pole at the bottom) and then turned off. The gel (gray box) has five numbered lanes where five
different samples of proteins (many copies of each kind of protein) were applied to the gel. (Lane 1,
molecular weight standards of known sizes; Lane 2, a mixture of three proteins of different sizes with
a being the largest and c being the smallest protein; Lane 3, protein a by itself; Lane 4, protein b by
itself; Lane 5 protein c by itself.) Notice that each group of the three proteins migrated the same
distance in the gel whether they were with other proteins (lane 2) or not (lanes 3-5). The molecular
weight standards are used to measure the relative sizes of the unknown proteins (a, b, and c).

Coomassie staining
Once protein bands have been separated by electrophoresis, they can be visualized using
different methods of in-gel detection. The most widely used method is Coomassie staining.
Several coomassie stain reagent recipes exist in the literature and use either the G-250
(“colloidal”) or R-250 form of the dye. The Coomassie dyes (R-250 and G-250) bind to proteins
through ionic interactions between dye sulfonic acid groups and positive protein amine groups as
well as through Van der Waals attractions. Coomassie R-250, the more commonly used of the
two, can detect as little as 0.1 ug of protein. Though less sensitive, Coomassie G-250 can be used
in place of the R-250 form to create a rapid and convenient staining procedure.
In acidic buffer conditions, coomassie dye binds to basic and hydrophobic residues of proteins,
changing from dull reddish-brown to intense blue. As with all staining methods, coomassie dye
reagents detect some proteins better than others based on their chemistry of action and
differences in protein composition. Thus, coomassie dye reagents can detect as few as 8-10
nanograms per band for some proteins and 25 nanograms per band for most proteins.
Coomassie dye staining is especially convenient because it involves a single, ready-to-use
reagent and does not permanently chemically modify the target proteins. An initial water wash step
is necessary to remove residual SDS, which interferes with dye-binding. Then stain reagent is
added, usually for about 1 hour; finally, a water (G-250) or simple methanol:acetic acid destaining
step (R-250) is used to wash away excess non-bound dye from the gel matrix. Because no
chemical modification occurs, excised protein bands can be completely destained and the proteins
recovered for analysis by mass spectrometry or sequencing.

36
 Figure 7.4: Photo of a Coomassie stained protein gel after the destaining step.

II-PRACTICE OBJECTIVES
 Describe the use of polyacrylamide gel electrophoresis for the analysis of complex protein
mixtures.
 Understand the different steps involved in performing a successful SDS-PAGE.
 Use SDS-PAGE to evaluate the successful purification of the mouse dihydrofolate
reducatse (DHFR) tagged with a 6xHis tag using the Ni-NTA protein purification kit.

III-MATERIAL

 Micropipettes of 1000, 200, 20 and 2 µl.

 Corresponding micropipette tips.
 50 ml tubes.
 25 ml sterile pipette.
 Eppendorf tubes.
 Dry heating block.
 Shaker agitator.
 Protein electrophoresis apparatus.
 PowerPack power source.
 Acrylamide-bisacrylamide.
 SDS 10%.
 APS 10%.
 TEMED.
 1.5 M Tris, pH 8.8.
 1.0 M Tris, pH 6.8.
 5x SDS Running Buffer (1 L).
 Coomassie Blue Stain.
 Isopropanol Fixing Solution.
 SDS sample loading buffer.
 10% (v/v) acetic acid.

IV-PROCEDURE

A- Polyacrylamide gel preparation:

1. Load samples and run gel @ 25 mA (2 gels run @ 50 mA) in 1x SDS Running Buffer.

2. Place gel in a plastic container. Cover with isopropanol fixing solution and shake at room
temperature. For 0.75 mm-thick gels, shake 10 to 15 min; for 1.5 mm thick gels, shake 30 to
60 min.

37
3. Pour off fixing solution. Cover with Coomassie blue staining solution and shake at RT for 2
hr.

4. Pour off staining solution. Wash gel with 10% acetic acid to destain, shaking at RT
overnight.

5. Place gel in a sealed plastic bag and take a photo.

B- Protein purification from frozen E. coli cells under denaturing conditions:

1. Load samples and run gel @ 25 mA (2 gels run @ 50 mA) in 1x SDS Running Buffer.

2. Place gel in a plastic container. Cover with isopropanol fixing solution and shake at room
temperature. For 0.75 mm-thick gels, shake 10 to 15 min; for 1.5 mm thick gels, shake 30 to
60 min.

3. Pour off fixing solution. Cover with Coomassie blue staining solution and shake at RT for 2
hr.

4. Pour off staining solution. Wash gel with 10% acetic acid to destain, shaking at RT.

4. Place gel in a sealed plastic bag and take a photo.

Questions:

1. Present the results including a photo of the gel and the different steps of the procedure.
2. Research the reasons that make the G-250 stain procedure easier and faster than then the
R-250 stain.
3. What is the protein marker made of?

PRACTICE 8

Western-blot.

I-INTRODUCTION

The term "blotting" refers to the transfer of biological samples from a gel to a membrane and their
subsequent detection on the surface of the membrane. Western blotting (also called
immunoblotting because an antibody is used to specifically detect its antigen) was introduced by

38
Towbin, et al. in 1979 and is now a routine technique for protein analysis. The specificity of the
antibody-antigen interaction enables a target protein to be identified in the midst of a complex
protein mixture. Western blotting can produce qualitative and semiquantitative data about that
protein.

The first step in a Western blotting procedure is to separate the macromolecules using gel
electrophoresis. After electrophoresis, the separated molecules are transferred or blotted onto a
second matrix, generally a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. Next, the
membrane is blocked to prevent any nonspecific binding of antibodies to the surface of the
membrane. Most commonly, the transferred protein is complexed with an enzyme-labeled
antibody as a probe. An appropriate substrate is then added to the enzyme and together they
produce a detectable product such as a chromogenic precipitate on the membrane for colorimetric
detection. The most sensitive detection methods use a chemiluminescent substrate that, when
combined with the enzyme, produces light as a byproduct. The light output can be captured using
film, a CCD camera or a phosphorimager that is designed for chemiluminescent detection.
Alternatively, fluorescently tagged antibodies can be used, which are directly detected with the aid
of a fluorescence imaging system. Whatever system is used, the intensity of the signal should
correlate with the abundance of the antigen on the membrane.

Detailed procedures for detection of a Western blot vary widely. One common variation involves
direct vs. indirect detection. With the direct detection method, the primary antibody that is used to
detect an antigen on the blot is labeled with an enzyme or fluorescent dye. This detection method
is not widely used as most researchers prefer the indirect detection method for a variety of
reasons. In the indirect detection method, a primary antibody is added first to bind to the antigen.
This is followed by a labeled secondary antibody that is directed against the primary antibody.
Labels include biotin, fluorescent probes such as fluorescein or rhodamine, and enzyme
conjugates such as horseradish peroxidase or alkaline phosphatase. The indirect method offers
many advantages over the direct method.

39
II-PRACTICE OBJECTIVES
 Describe the use of western blot for the detection of a protein of interest in a complex
mixture.
 Understand the different steps involved in performing a successful Western blot.
 Use the antibody specific to the DHFR protein in order to detect the presence of this protein
in a complex extract or in the nickel column purified fraction.

III-MATERIAL

 Micropipettes of 1000, 200, 20 and 2 µl.

 Corresponding micropipette tips.
40
  Eppendorf tubes.
 Shaker agitator.
 Membrane transfer apparatus.
 Nitrocellulose membranes.
 Primary antibody.
 Secondary antibody.
 Blocking agent.
 ECL western blot kit.
 Film cassette.
 Film.
 Dark room.
 Film developing and fixing solutions.
 Transfer buffer.
 Blotting buffer.

IV-PROCEDURE

1. Run SDS-PAGE.

2. Wet membrane in H2O. Soak membrane in transfer buffer for 10 min.

3. Set up transfer from the gel to a nylon membrane in transfer buffer.

4. Place “transfer sandwich” in semi-dry transfer chamber. Run at 23 V for 30 min for 0.75 and
1.0 mm gels or 40 min for 1.5 mm gel.

5. Block blot by soaking in blotting buffer for 1 hr with shaking. Alternatively, blocking can be
done with as much as 10% milk and 0.5% Tween 20 to reduce background.

6. To 10 ml blocking solution, add primary antibody at proper dilution. Incubate the membrane
for 1 hr with shaking. Alternatively, incubation with 1º Antibody can be done ON @ 4 ºC.

7. Wash 2x briefly with PBS-T, then wash 3x with PBS-T for 5 min.

8. To 10 ml PBS-T, add secondary antibody at proper dilution. Incubate the membrane for 1 hr
with shaking.

9. Wash 2x briefly with PBS-T, then wash 3x with PBS-T for 5 min.

10. Detection by ECL according to the manufacturer instructions. Expose blot to film for 15 sec
– 5 min.

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