UDBB1224/UDEE2124 Principles of Biotechnology

EXPERIMENT 1:

Title: Agarose Gel Electrophoresis

Introduction:
Gel electrophoresis is a standard method used to separate, identify and purify DNA fragments.
Two types of gels are commonly used: agarose and polyacrylamide gels. Electrophoresed DNA
can be visualized by staining the agarose gel with dyes such as ethidium bromide (EtBr), SYBR
Green and GelRed. Factors that may influence the rate of DNA migration in the agarose gel are
(i) the size and conformation of the DNA, (ii) gel concentration, (iii) composition of the
electrophoresis buffer, (iv) voltage, and (v) the presence of DNA intercalating dyes.

Materials:
UV transilluminator
Electrophoresis set and power pack
Agarose powder
DNA loading dye
DNA ladder
DNA sample
1× TAE or 1× TBE
DNA staining solution

Methods:
(I) Agarose Gel Preparation:

1. Add the appropriate amount of agarose powder and volume of agarose gel running buffer
(1 × TBE or TAE buffer) in a flask to prepare a 25 ml 0.8% (w/v) agarose gel.
2. Heat the slurry in a microwave oven until the agarose completely dissolves in the buffer.
3. Cool the gel solution until about 50°C, add in GelRed nucleic acid gel stain, swirl the gel
solution before pouring into the gel cast. The thickness of the gel should be ~5 mm.
4. Position the comb ~2 mm above the bottom of the gel cast. Do not move or jar the gel
cast/comb while the gel is being set.
5. After the gel is completely set (~20 min), carefully remove the comb. Mount the gel in
the electrophoresis tank.
6. Fill the tank with the agarose gel running buffer (should be the same buffer used to
prepare the agarose gel in Step 1) to cover the gel.

Note: The gel can be kept in the buffer until you are ready to continue.

(II) DNA Loading:

1. Add gel loading dye to each DNA sample and briefly mix.
2. Load DNA into the wells of the gel. Use a fresh tip for each new sample. Be careful to
expel any air from the micropipette tip end before loading gel. Take care not to punch tip
through the bottom of the gel.
3. Load a molecular size standard/marker in a separate well.

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UDBB1224/UDEE2124 Principles of Biotechnology

(III) Electrophoresis:

1. Place the lid on the electrophoresis apparatus. Connect the power cords between the unit
and the power supply. Make sure that the power cords are not reversed!
2. Turn on the power supply and adjust the voltage. The migration of DNA through agarose
gel is voltage-dependent.
3. Monitor the movement of the bromophenol blue band until it has migrated near the end
of the gel.
4. Turn off the power, disconnect the power cords and remove the lid from the unit.

(IV) Gel Staining and Viewing:

1. View the gel under an ultraviolet transilluminator.

2. If the agarose gel in not pre-stained with nucleic acid gel stain, after electrophoresis, the
gel has to be stained with nucleic acid solution for 5 to 10 min before viewing under
ultraviolet transilluminator

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UDBB1224/UDEE2124 Principles of Biotechnology

EXPERIMENT 2:

Title: Extraction of Plasmid DNA

Introduction:

Plasmids
Plasmids are relatively small, double-stranded, closed-circular DNA molecules that exist apart
from the chromosomes of their hosts. Plasmids are present in a wide variety of bacterial and
fungal species. Naturally occurring plasmids carry one or more genes. For example, some
plasmids carry genes which confer resistance to certain antibiotics. Some may carry genes that
direct the synthesis of enzymes that aid in the production of bacterial poisons or antibiotics.

From the viewpoint of the genetic engineer, however, the most important property of plasmids is
that they bear a special region of DNA called an origin of replication, or more simply an origin.
This region allows the plasmid to multiply within and semi-independently of its host. It will
replicate its own DNA as well as any passenger DNA that may be attached to it, producing many
copies of the recombinant molecule.

Plasmid is now widely used as a cloning vector. In a typical cloning experiment, the circular
plasmid DNA is cut open with restriction endonuclease(s) at its multiple cloning sites. This
converts the circular molecule into a linear form. Then, a foreign DNA fragment with the
compatible ends is inserted at the multiple cloning sites with the help of the enzyme DNA ligase.
The ligase creates a circular molecule containing both the plasmid and its passenger producing a
recombinant DNA molecule. Once inside a suitable host, the plasmid produces many copies of
itself as the bacteria themselves grow and reproduce.

Extraction and Purification of Plasmid DNA

Many methods have been developed to purify plasmids from bacteria and these methods
invariably involved three steps:
 Growth of bacterial culture
 Harvesting and lysis of the bacteria
 Purification of the plasmid DNA

For many years, equilibrium centrifugation in CsCl-ethidium bromide gradients was the method
of choice to prepare large amounts of plasmid DNA. However, this process is time consuming
and requires expensive equipment and reagents. Nowadays, less expensive and faster methods
are available to purify smaller plasmids (< 15 kb). These methods rely on differential
precipitation, ion-exchange chromatography, or gel filtration to separate plasmid DNA from
cellular nucleic acids.

The alkaline lysis method is by far the most popular because of its simplicity, inexpensive, and
reproducibility. A variety of kits for plasmid purification are also available from commercial
vendors. These kits consist of disposable chromatography columns that are used for batch
absorption and elution of plasmid DNA. However, this convenience comes at a price. In this
practical, students shall perform plasmid extraction from bacterial culture using the alkaline lysis

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UDBB1224/UDEE2124 Principles of Biotechnology

method. The quality and the quantity of the extracted plasmid will then be determined. The
integrity of the extracted plasmids can be visualized in agarose gel electrophoresis. Plasmids are
usually resolved in agarose gels with appropriate gel concentrations (0.7% - 1.0%w/v).

Materials
1. Escherichia coli JM109 or DH5 strains containing an appropriate plasmid
2. Alkaline lysis solution I
(50 mM glucose; 25 mM Tris-Cl, pH 8.0; 10 mM EDTA, pH 8.0)
3. Alkaline lysis solution II
[0.2 N NaOH, 1% (w/v) SDS, solution II should be prepared freshly and used at room
temperature)
4. Alkaline lysis solution III
(3 M potassium, 5 M acetate)
5. 95% (v/v) ethanol
6. 70% (v/v) ethanol
7. Phenol:chloroform (1:1, v/v)
8. DNase free RNase
9. TE buffer
10. Microfuge tubes
11. Micropipettes and tips

Solution I: 25 mM Tris-Cl (pH 8.0)

10 mM EDTA (pH 8.0)
50 mM glucose

Solution II: 0.2 M NaOH (prepared fresh from stock solutions of 10 N NaOH)
1.0% (w/v) SDS

Solution III: 5 M potassium acetate 60.0 ml

Glacial acetic acid 11.5 ml
Distilled water 28.5 ml

Methods:

Preparation of Cell
1. Inoculate a single colony containing the plasmid DNA of interest in 5 ml of LB medium
and 50 g/ml of appropriate antibiotics (e.g., ampicillin), depending on the specific
antibiotic-resistant gene carried by the specific plasmid. Culture the bacteria at 37C
overnight with shaking at 200 rpm.
2. Transfer the overnight culture into microcentrifuge tubes (1.5 ml per tube), centrifuge at
maximum speed for 2 min, and then discard the supernatant.

Lysis of Cells
1. Resuspend the bacterial pellet in 150 l of ice-cold alkaline lysis solution I by vigorous
vortexing or by pipetting (make sure that the bacterial pellet is completely dispersed

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UDBB1224/UDEE2124 Principles of Biotechnology

in Alkaline lysis solution I. Vortexing the microfuge tubes simultaneously with their
bases touching increases the rate and efficiency with which the bacterial pellets are
resuspended)
2. Add 200 l of freshly prepared Alkaline lysis solution II to each bacterial suspension.
Close the tube tightly, and mix the contents by inverting the tube rapidly five times. DO
NOT vortex! Store the tube on ice for 5 min
3. Add 150 l of ice-cold Alkaline lysis solution III. Close the tube and disperse Alkaline
lysis solution III through the viscous bacterial lysate by inverting the tube several times.
Store the tube on ice for 5 min.
4. Centrifuge the bacterial lysate at maximum speed (13 000 rpm) for 5 min and carefully
transfer the supernatant to a fresh tube.
5. (Optional) Add an equal volume of TE-saturated phenol:chloroform. Mix the organic and
aqueous phases by vortexing for 1 min and then centrifuge the emulsion at maximum
speed for 10 min. Transfer the aqueous upper layer to a fresh tube

Recovery of Plasmid DNA

1. Precipitate nucleic acids from the supernatant by adding 2 volumes of 100% ethanol (or 1
volume of propanol) at room temperature. Mix the solution by vortexing and then allow
the mixture to stand for 2 min at room temperature.
2. Collect the precipitated nucleic acids by centrifugation at maximum speed for 10 min.
Carefully decant the supernatant, add 1 ml of 70% ethanol to the pellet and invert the
closed tube several times. Recover the DNA by centrifugation at maximum speed for 5
min.
3. Remove all of the ethanol by gentle aspiration and dry the plasmid DNA under vacuum
for 15 min or store the open tube at room temperature until the ethanol has evaporated
and no fluid is visible in the tube.
4. Dissolve the plasmid DNA in 50 l of TE (pH 8.0) buffer or sterile deionized water and
store the DNA solution at -20C until use.

Reference:
1. Sambrook, J., Russell, D. W., Sambrook, J. (2001). Molecular Cloning: A Laboratory
Manual. Cold Spring Harbor Laboratory

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UDBB1224/UDEE2124 Principles of Biotechnology

EXPERIMENT 3:

Title: Analysis of Extracted Plasmid DNA

A complete understanding of the biochemical functions of DNA requires a clear picture of its
structure and physical characteristics. DNA has a significant absorbance in the UV range due to
the presence of the aromatic bases – adenine, guanine, cytosine and thymine. This allows useful
observation to be carried out as the structural changes such as helix unwinding which affects the
extent of absorption.

Absorption measurements (spectrophotometric method) can also be used as an indication of the

DNA purity and quantity. Another common method of characterizing nucleic acids (DNA and
RNA) is by using agarose gel electrophoresis. The DNA (or RNA) samples are visualized under
the UV transilluminator. This method is useful in determining the integrity of the extracted
nucleic acids.

The amount of DNA in the sample can be estimated by taking absorbance readings at 260 nm
and 280 nm. DNA absorbs maximally at the wavelength of 260 nm. On the other hand, proteins
absorb maximally at 280 nm.

An OD reading of 1.0 at 260 nm corresponds to a concentration of 50 µl/ml of dsDNA.

dsDNA concentration (µg/ml) = A260 x conversion factor x dilution factor

The ratio between readings at 260 nm and 280 nm provides an estimate of the purity of the
sample. Pure preparations of nucleic acids have A260/A280 ratios of 1.8 - 2.0. Protein or other
contaminants (e.g. phenol) will yield the ratio of < 1.8.

Materials:

UV transilluminator
Spectrophotometer
Cuvettes
Electrophoresis set and power pack
DNA loading dye
1× TAE or 1× TBE
0.7% - 1.0% (w/v) agarose
DNA staining solution

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UDBB1224/UDEE2124 Principles of Biotechnology

Methods:

(I) Spectrophotometric Determination of DNA

DNA can be quantified using a spectrophotometer. In your experiment, the plasmid DNA will be
quantified using the Thermo Scientific NanoDrop™ 1000 Spectrophotometer that measures 1 ul
samples with high accuracy and reproducibility.

(II) Agarose Gel Electrophoresis

The extracted plasmid DNA of Experiment 2 will be electrophoresed using 0.8% agarose gel.

For the detailed experimental procedure of agarose gel electrophoresis, please refer to the
Experiment I.

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UDBB1224/UDEE2124 Principles of Biotechnology

EXPERIMENT 4:

Title: Restriction Enzyme Digestions

Introduction:

A restriction map is a description of restriction endonuclease cleavage sites within a piece of

DNA. Generating such a map is usually the first step in characterizing an unknown DNA, and a
prerequisite to manipulating it for other purposes. Typically, restriction enzymes that cleave
DNA infrequently (e.g. those with 6 bp recognition sites) and are relatively inexpensive are used
to produce a map. The most straightforward method for restriction mapping is to digest samples
of the plasmid with a set of individual enzymes (single digestion), and with pairs of those
enzymes (double digestion). One unit of restriction endonuclease corresponds to the amount of
enzyme required to completely digest 1 µg of  DNA in 1 hour of incubation under optimal
assay conditions.

The resulting restriction fragment after restriction enzyme digestions will be analyzed by gel
electrophoresis. The concentration used for agarose gel preparation depends on the size of DNA
fragments to be analyzed. Most of the DNA fragments are resolved in agarose gels with
concentrations between 0.7% - 1.0%. Separation of fragments smaller than 0.5 kb may require
gel concentration of 1.5-2.0%, whereas separation of DNA fragments between 10 - 30 kb may be
performed using 0.5% agarose gel.

Materials:
Plasmids pBR322, and/or any other appropriate plasmids
Restriction enzymes and their corresponding buffers
37C water bath
Agarose gel electrophoresis apparatus
UV transilluminator
DNA loading dye
DNA staining solution
Microcentrifuge

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UDBB1224/UDEE2124 Principles of Biotechnology

Methods:

1. Label microcentrifuge tube that will be added with corresponding restriction enzymes.
2. Prepare reaction mixes as follows:

IMPORTANT: Use a fresh pipette tip for each reagent!

Three different restriction enzymes will be provided. You are required to prepare the following
single restriction digestion mixtures:

Reagent Single Digestion (µl)

DNA 5
10x Buffer 2
RE1 or RE2 1
or RE3
Top-up to 20 μl with dH2O

3. Prepare combinations of the following double digestions:

(i) RE1 and RE2
(ii) RE1 and RE3
(iii) RE2 and RE3

Double Digestion
(µl)
DNA 5
10x Buffer 2
RE1 and RE2 1 μl of each RE
or
RE1 and RE3
or
RE2 and RE3
Top-up to 20 μl with dH2O

4. Quick spin each tube contents to collect samples at the bottom of each tube.
5. Incubate all reaction mixtures at 37°C for 1-2 hour.
6. Add 1-2 µl of gel loading buffer (dye) to 5 µl of the reaction mixture.
7. Load each sample into a well of the agarose gel. Appropriate DNA molecular weight
marker will be loaded in a separate well.
8. Electrophorese the 1% agarose gel for about 45 min at 90 V.
9. Visualize the DNA fragments under the UV transilluminator.
10. Determine the size of each separated DNA fragment.
11. From the DNA fragment sizes, reconstruct the plasmid map.

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UDBB1224/UDEE2124 Principles of Biotechnology

EXPERIMENT 5:

Title: Polymerase Chain Reaction (PCR)

Introduction
Polymerase Chain Reaction (PCR), invented by Kary B. Mullis, at the Cetus Corporation, who
was awarded the 1993 Nobel Prize for chemistry for PCR, is a technique to exponentially
amplify in vitro a small quantity of a specific nucleotide sequence.

A PCR reaction consists of the template DNA to be amplified, a pair of oligonucleotide primers
with sequence flanking the region to be amplified, DNA Taq polymerase, the four
deoxynucleoside triphosphates (dNTP), magnesium, and appropriate buffer and salts. The
reaction is cycled involving template denaturation, primer annealing, and the extension of the
annealed primers by DNA polymerase until enough copies are made for further analysis. During
the PCR process, the product of 1 cycle serves as template in the next cycle. Amplification of
template progress at a rate of 2n, where n is equal to the number of cycles.

PCR is a very powerful technique and is used in wide range of applications. To cite only a few, it
is used to examine biological evidence in forensic cases, to identify contaminating
microorganisms in food, to diagnose genetic diseases, to map genes to specific chromosome
segments, to detect the presence of a specific DNA in a particular sample, etc.

In this practical you will use the plasmid DNA extracted from Experiment 2 as template to
amplify the GFP (green fluorescent protein) gene. The PCR product can be detected by
performing 1.5% agarose gel electrophoresis.

Materials:
 Sterile H2O
 25 mM or 50 mM MgCl2
 PCR microtubes
 Micropipettes & tips
 10 × PCR buffer
 10 mM dNTP mix
 10 μM oligonucleotides primers 1 and 2
 Template DNA
 Taq DNA polymerase (5 U / µl)
 Automated PCR thermal cycler
 Agarose gel electrophoresis apparatus

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UDBB1224/UDEE2124 Principles of Biotechnology

Methods

Preparation of Reaction Mixture

To perform several parallel reactions, prepare a master mix containing all the PCR components
except the template DNA in a single tube, which can then be aliquoted into individual tubes.
Template DNA solutions are then added. This method of setting reactions minimizes the
possibility of pipetting errors and saves time by reducing the number of reagent transfers.

Reaction Mixture Set Up

1. Briefly centrifuge all solutions after thawing.
2. Label your PCR reaction tubes and a sterile 1.5 ml microfuge tube (for master mix
preparation).
3. Calculate the quantity of each individual PCR component and prepare a master mix solution
for 3 reactions in the sterile 1.5 ml microfuge tube as follow:

Reagent Final Quantity for 1 Quantity for master

concentration reaction mixture (µl) mix (µl)
Sterile deionized water -
10 × Taq buffer 1
dNTP mix 0.2 mM of each
Primer I 0.4 µM
Primer II 0.4 µM
Taq DNA Polymerase 1 U per reaction
MgCl2 2 mM
Template DNA 50 – 100 ng
Total volume 25

4. Briefly centrifuge the reaction mixture to collect all drops from the wall of the tube. Aliquot
the appropriate amount of master mix to each reaction tube.
5. Add the DNA template into the labeled PCR tubes.
6. Overlay the sample with a drop of mineral oil. This step may be omitted if the thermal cycler
is equipped with a heated lid.
7. Place samples in a thermocycler and run the PCR using the following PCR thermal profile:

Cycling steps Temp/Time Number of cycles

Initial denaturation 95C/3 min 1
Denaturation 95C/30 s
Primer annealing 53C/30 s 30
Extension 72C/1 min
Final Extension 72C/5 min 1

8. After the PCR is completed, analyse the PCR product by agarose gel electrophoresis.

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