STB 2163

Recombinant DNA
Technology
Practical Manual

Facilitators/Editors

Prof. Dr Mohd. Hasnain Md. Hussain

Assoc. Prof. Dr Hairul Azman Roslan
Dr Rosmawati Saat
Dr Simon Ngieng

Faculty of Resource Science

and Technology
Universiti Malaysia Sarawak

Semester 2
Session 2023-2024
Recombinant DNA Technology Practical Manual STB2163

Introduction
Modern Biotechnology is a field that covers Genetic Engineering Technology
and has become a major part in most biological research. Even though DNA-
based Biotechnology is relatively new in comparison to many fundamental
biological science disciplines it is now a major contributor to our knowledge of
the living system. We have entered a new era of studies in which we can
study the structure and function of individual genes as a result of
advancement in methods for isolating, manipulating and amplifying identifiable
sequences of DNA. These approaches and associated experimental
techniques are collectively known as Recombinant DNA Technology, and
represent powerful ways of studying the biological systems.

Although genetic recombination does occur naturally, Recombinant DNA

Technology allows direct engineering of new organisms and biomolecules.
This involves the intentional recombination of genes from different sources. In
addition to being a common tool for basic research, Recombinant DNA
Technology has numerous industrial applications. The benefits of such
application can be seen in the development of diagnostic and therapeutic
products; creation of hardier value-added crops and commercially-viable
livestock; and super microorganisms amenable for industrial and
environmental applications.

This practical class module is designed to provide a hands-on experience in

basic Recombinant DNA techniques. These include basic protocols in
extraction of genomic and plasmid DNA from different organisms, restriction
endonuclease analysis, agarose gel electrophoresis, cloning, and simple gene
cloning.

Objective
The objectives of this practical class module are to allow students to:

1) Apply standard skills in operating common Molecular Biology laboratory

equipment.
2) Apply the principles of Recombinant DNA techniques in the laboratory
environment.

Course Assessment
This course is assessed on the basis of two examination (mid-semester and
final exam) and lab reports.
Assignments & quizzes 20%
Laboratory reports 20%
Mid Semester Exam 20%
Final Examination 40%
Overall 100%
Recombinant DNA Technology Practical Manual STB2163

Guideline on Laboratory Report Writing

The writing of the practical and mini project reports will follow the
journal
format outlined as shown below: -

Title
Introduction
Objective
Materials and methods
Results and discussion
Conclusion (if any)
References (refer to examples below)

Examples of references: -
Sambrook, J., Fritsh, E.F., & Maniatis, T. (1989). Molecular Cloning: A
Laboratory Manual (2nd ed.). Cold Spring Habor Laboratory Press. New York.

Zyskind, J.W., & Bernstein, S.I. (1992). Recombinant DNA Laboratory

Manual. Academic Press Inc. San Diego, Carlifornia, USA.

Brown, T.A. (2006). Gene Cloning and DNA Analysis: An Introduction (5th
ed.). Blackwell Publishing. UK.
Practical 2A STB2163 Recombinant DNA Technology Semester 2, Session 2023/2024

PRACTICAL 2A:
MINI‐PREP ISOLATION OF DOUBLE‐STRANDED PLASMID DNA
FROM BACTERIAL CULTURE

Introduction:
The mini preparation of plasmid DNA, or miniprep, is a small-scale isolation of plasmid DNA
from bacteria. Two main approaches have been used for this isolation; the first and more
traditional technique of alkaline lysis method 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. We will use the
traditional method for the miniprep and you will have an opportunity to use the spin-columns
in other isolation procedures.

In brief, bacterial cells containing the desired plasmid are harvested from liquid bacterial
culture by centrifugation. Suspension of bacteria is made in isotonic solution which is
subsequently subjected to lysis by an alkaline solution containing a detergent, Sodium dodecyl
sulfate (SDS), and an alkali, sodium hydroxide (NaOH). While sodium dodecyl sulfate serves to
lyse cells and denature proteins, alkaline condition denatures genomic DNA, plasmid DNA and
proteins. Lysed cell mixture is further neutralized by potassium acetate (pH 5.2). This resulted in
the renaturation of plasmid and genomic DNA. Since plasmid DNA is covalently closed, it will
reanneals while genomic DNA form precipitate. The precipitated plasmid DNA is separated by
high speed centrifugation. Plasmid from the supernatant is recovered by precipitation using
isopropanol or ethanol.

Objective:
To get students to understand the biochemical and molecular effects of each reagent used in
the miniprep plasmid DNA protocol as well as the difference in mobility between a DNA
fragment and an intact plasmid when analyzed on agarose gel.

1
Practical 2A STB2163 Recombinant DNA Technology Semester 2, Session 2023/2024

EXPERIMENT 2A:

MINI PREP ISOLATION OF PLASMID DNA FROM BACTERIA USING ALKALINE LYSIS
METHOD

Materials:
Overnight bacterial cell culture containing cloned plasmid
Eppendorf tubes
Cold absolute ethanol
70% ethanol

Solution I: (keep on ice)

50 mM Glucose
10 mM EDTA, PH 8
25 mM Tris-HCl, pH 8

Solution II:
0.2 N NaOH
1% SDS

Solution III: (keep on ice)

5 M KAc (Potassium acetate), glacial acetic acid

Methods:

1. Harvest the bacterial cells from a 2 mL overnight culture by transferring the culture
into a 2 mL microcentrifuge tube and centrifuging at 8000 rpm for 2 minutes at
room temperature.

2. Remove the supernatant (culture media) carefully, and recentrifuge the pellet for 1
minute. Remove completely any traces of liquid media from the tube.

3. Resuspend the cell pellet using 100 µl of Solution I. Resuspend by vortexing briefly
for 10 seconds.

4. Add 200 µl of Solution II to the cell suspension and mix gently by inverting the tube
5X. Leave the tube on ice and allow the lysis reaction to occur for 5 minutes. Do
NOT Exceed 5 minutes. A clear viscous liquid will be observed.

5. Add 150 µl of Solution III and mix by inverting the tube 10X. A white precipitate will
be observed. Pellet the precipitate by centrifuging at 13,000 rpm for 5 minutes.
Carefully transfer the supernatant (containing plasmid DNA) into a sterile 1.5 ml
microcentrifuge tube.
2
Practical 2A STB2163 Recombinant DNA Technology Semester 2, Session 2023/2024

6. Precipitate the DNA by adding 2 volume of cold absolute ethanol. Mix the content
gently by inverting the tube at least 10X.

7. Pellet the DNA by centrifuging at 13,000 rpm for 5 minutes at room temperature.
Discard the supernatant and wash the pellet with 500 µl of 70% ethanol, and
recentrifuge at 13,000 rpm for 2 minutes.

8. Discard as much of the supernatant as possible and allow the DNA pellet to air dry
for 15 minutes at room temperature. Resuspend and dissolve the DNA pellet in 50
µl of TE buffer.

9. Keep and store the plasmid DNA sample at -20 °C for further analysis using agarose
gel electrophoresis (AGE).

3
Practical 2B STB2163 Recombinant DNA Technology Semester 2, Session 2023/2024

PRACTICAL 2B:
RESTRICTION ANALYSIS OF PLASMID USING COMMON RESTRICTION ENZYMES &
AGAROSE GEL ELECTROPHORESIS (AGE) ANALYSIS.

Introduction:
Interpreting the gels showing plasmid DNA is not always straightforward and requires practice
and the “dirtier” miniprep DNA can further complicate the interpretation.

Movement of DNA Fragments

Pieces of DNA, for example DNA fragments cut by restriction enzymes, separate in an agarose
gel according to size. The smaller pieces travel the fastest and the farthest through the gel. The
larger pieces have more difficulty moving through the gel matrix and thus move more slowly
through the gel. However, the movement of DNA fragments in the gel is logarithmic and not
arithmetic. This means that the smaller (lighter) fragments move faster than one would expect
and the larger (heavier) fragments move even slower than one would expect if the relationship
were arithmetic. When we use a standard curve of known fragment sizes to determine the size
of fragments of unknown length, the distance migrated is plotted on an arithmetic scale (in
millimeters or centimeters traveled). But the size or mass of the fragment is plotted on a log10
scale.

Fragment Resolution
There are limits to the ability of agarose to separate DNA fragments of different sizes. When
two fragments are close together in size, they may appear as one band in the gel rather than
two. For example; if you expect 6 DNA fragments and thus 6 bands on a gel but you see only 5
bands, then it is likely that two of the fragments have not resolved in the gel. It is termed a
doublet when two fragments appear as one band.

Conformations of Uncut Plasmid DNA

We will analyze DNA that has been cut with restriction enzymes by determining fragment sizes
using agarose gel electrophoresis. However, uncut plasmid DNA has several distinct
conformations that can be identified when the uncut plasmid is electrophoresed in an agarose
gel.

a) Supercoiled DNA (Form I), is the fastest conformation of the uncut plasmid. The enzyme
DNA gyrase introduces these extra twists into chromosomal and plasmid DNA of
bacteria. The bacteria use the superhelical tension to assist in processes like replication
and transcription. The isolated supercoiled (sc) plasmid DNA is wound up into a compact
structure. Imagine taking a circle of string and rolling it around in your hands until it
forms a little ball. Because of its compact shape, sc DNA is the fastest moving
conformation in the gel.

4
Practical 2B STB2163 Recombinant DNA Technology Semester 2, Session 2023/2024

b) Nicked Circle DNA (Form II) is also called relaxed circle. In bacteria, the enzyme
topoisomerase I will nick one strand of the helix so that DNA polymerase has access to
the DNA for replication. Once one of the strands has been cut, the superhelical tension
relaxes and the tightly-wound ball becomes a floppy circle. A nick can also occur during
isolation of the plasmid because of enzyme activity or mechanical shearing of the DNA.
Nicked circle (nc) is the slowest conformation of uncut DNA.

c) Linear DNA (Form III) is produced when a restriction enzyme cuts a plasmid at only one
site. Both strands of the helix are cut at the same place. Linear DNA can occur because
of endonuclease contamination of the isolated plasmid or because of harsh treatment.
On a gel, the linear DNA will run between the sc and nc conformations (possibly closer
to the sc band).

These are the three main conformations of single plasmid molecules, or monomers.However,
under certain conditions, two plasmid molecules can join to form a dimer. Since the dimer form
is twice as large as the monomer, it will run higher in the gel. It is possible to have sc, nc, and
linear forms of the dimer. As with the monomeric plasmid, the sc will be the fastest
conformation and the nc will be the slowest. It is also possible for more than two plasmid
molecules to join and create high molecular weight multimers. It is usually difficult to
distinguish conformations of multimers.

Objective:
The objective of the experiments is to get the students to understand the differences in terms
of mobility between a DNA fragment and an intact plasmid. At the same time the students will
be able to know and recognize the major differences between the main conformations of uncut
plasmid DNA and be able to recognize them on a gel.

5
Practical 2B STB2163 Recombinant DNA Technology Semester 2, Session 2023/2024

EXPERIMENT 2B
RESTRICTION ANALYSIS OF PLASMID USING COMMON RESTRICTION ENZYMES &
AGAROSE GEL ELECTROPHORESIS (AGE) ANALYSIS.

Materials:
10X RE Buffer
Plasmid DNA
Restriction endonuclease
Sterile ddH2O
Eppendorf tubes

Methods:
1. Prepare the reaction for restriction digestion by adding the following reagents in the
order listed to an Eppendorf tube:

Sterile ddH20 z – (w+x+y) µL

10X assay buffer one-tenth of total volume (w) w µL
Plasmid DNA, x µL
Restriction enzyme*, y µL
(1-10 units per µg of plasmid DNA)
Total volume, z µL

2. *If desired, more than one enzyme can be included if both enzymes are active in the
same buffer and the same incubation temperature.

3. Note: The volume of the reaction depends on the amount and size of the DNA being
digested. Larger DNAs should be digested in larger total volumes (between 50-100 µL),
as should greater amounts of DNA. Refer to the vendor's catalogue for the chart of
enzyme activity in a range of salt concentrations to choose the appropriate assay buffer.

4. Mix well by gently pipetting the reaction mixture and incubate the reaction at the
appropriate temperature (typically 37oC) for 1-3 hours, in a dry heater block (or oven).

5. Inactivate the enzyme(s) by heating at 65oC for 10 minutes or by phenol extraction.

6. Prior to use in further protocols such as dephosphorylation or ligation, an aliquot of the

digestion mixture should be analyzed by agarose gel electrophoresis analysis versus the
non-digested DNA and a DNA molecular size marker, if necessary. The DNA should be
gel purified or ethanol precipitated before use in the subsequent cloning experiments.

6
Practical 2C STB2163 Recombinant DNA Technology Semester 2, Session 2023/2024

PRACTICAL 2C:
THE PREPARATION OF COMPETENT Escherichia coli CELLS.

Introduction:

Bacteria are able to take up DNA from their environment (exogenous DNA) in three ways;
conjugation, transformation, and transduction. Only transformation is the direct uptake of
DNA, since conjugation requires cell-cell contact via a sex pilus and transduction requires a
bacteriophage intermediary to transfer DNA from one cell to another.

For a bacterial cell to take up DNA from its surroundings, it must be in a special physiological
state called competence. Experiments by Frederich Griffith in 1929 using competent
Streptococcus (now Enterococcus) pneumoniae were instrumental in showing that DNA was
the transforming principle – the genetic material.

Natural competence is highly regulated in bacteria, and the factors leading to competence
vary among genera. For some genera, only a portion of the population is competent at any
time; for others, the entire population gains competence. A series of competence proteins is
produced, which have some homology but differ in the Gram negative and the Gram
positive bacteria.

Artificial competence is not encoded in the cell's genes. Instead it is a laboratory procedure
in which cells are passively made permeable to DNA, using conditions that do not normally
occur in nature. These standard procedures are comparatively easy and simple, and can be
used to genetically engineer bacteria. Chilling bacterial cells in the presence of divalent
cations such as CaCl2 or MgCl2 prepares the bacterial cell walls to become permeable to
plasmid DNA. However, transformation efficiency is, in general, low. Only a portion of the
cells become competent and a fraction of those are successful in taking up DNA.

This protocol reproducibly generates competent cultures of E. coli that yield 1 x 108 to
3 x 108 transformed colonies/µg of plasmid DNA.

Objective:
The objectives of the experiments are to get the students to be familiarize with how cells
are made competent which is the primary step for transformation.

7
Practical 2C STB2163 Recombinant DNA Technology Semester 2, Session 2023/2024

EXPERIMENT 2C
THE METHOD TO PREPARE E. coli COMPETENT CELLS.

Materials:

Buffers & Solutions

Prepare TSS solution:
Autoclave individually: 85 mL LB, 10 g PEG in 5 mL autoclaved distilled water,
5 mL DMSO, 2 mL 1 M MgCl2. Allow those to cool, then combine,
filter sterilize and fridge

Media
1. Luria bertani (LB) for initial culture growth.

Centrifuge & Rotors

1. Centrifuge machine.

Special Equipments (for competent cells prep and bacterial transformation)

1. Liquid nitrogen
2. Polypropylene tubes
3. Shaking incubator (18˚C)
4. Water bath at 42˚C
5. Chilled microfuge tubes

Reference
OpenWetWare. Competent cells preparation: Large and Small scales.
https://openwetware.org/wiki/Making_Competent_Cells

Inoue H., Nojima H., and Okayama H. (1990). High efficiency transformation of
Escherichia coli with plasmids. Gene, 96, 23-28.

8
Practical 2C STB2163 Recombinant DNA Technology Semester 2, Session 2023/2024

Methods:

Step 1: Growing Bacterial Cultures (Day 1)

1. Pick a single bacterial colony (2-3 mm in diameter) from a plate that has been
incubated for 16‐20 hours at 37˚C.
2. Transfer colony into 15 mL of LB broth in at 50mL flask.
3. Incubate culture for 16‐20 hours at 37˚C with vigorous shaking (250‐300rpm).

Step 2: Harvesting Cells and Freezing Competent Cells (Day 2)

1. Inoculate a flask of 5 mL LB broth with 1 mL of culture from Step 1 (starter culture).

2. Incubate flask at 37˚C with moderate shaking.
3. Read the OD600 of all three cultures. Continue to monitor every 15 mins until
reading is at 0.5-0.6.
4. Transfer the culture vessel to ice water bath for 5 min.
5. Transfer 1 ml of the culture into 2 microfuge tubes and harvest cells by
centrifugation at 10000 rpm for 3 minutes at RT.
6. Pour off medium and dry the tube (inverted) on paper towels for 1 min
(use vacuum aspirator or gently tap on the paper towel to remove any drops of
remaining medium adhered to the walls of the centrifuge tube).
7. Resuspend the cells gently (by swirling) in 90 uL of ice‐cold (0˚C) TSS buffer.
(You can use immediately after this step or keep in -20C)
8. Snap freeze competent cells in liquid nitrogen (store stock at ‐80˚C).
9. When needed, remove tube of competent cells from freezer, thaw in hand, use
immediately.

Note.
Prechill all materials such as rotor, tubes and TSS solution for the preparation of competent cells.