School of Doctoral Studies (European Union) Journal
128
2010
Cloning DNA through the use of Recombinant DNA
Technology
Erik Rotenhoffen (MSc)
Candidate to PhD in Science at the School of Doctoral Studies EU
Square de Meeus 37-4th Floor 1000 Brussels, Belgium
email: erotenhoffen.gen@dsc.sds.eu
Abstract
Recombinant DNA technology was used to clone specific DNA fragments. Before the technology was used, a basic
understanding of the parts and details of the procedure were investigated. It was found that the larger the genome of an
organism, the greater its overall complexity relative to other living organisms. This was discovered through the comparison
of various genome sizes coming from known specimen. Gel electrophoresis technology was utilized to compare the relative
sizes of the DNA. The gels were run from left to right with increasing level of complexity. The samples were, from left to
right, plasmid DNA, lambda bacteriophage DNA, calf thymus DNA, and calf kidney DNA. As the complexity of the DNA
increased, the number of bands in the lane increased to the point that the Eukaryotic DNA found in the calf thymus and
kidney were smears. The gel displayed identical smearing and satellites for the calf kidney and thymus DNA, proving that the
DNA is the same even though they are from different part of the organism. Through DNA fingerprinting, DNA fragments
were determined that matched a specific pattern after being exposed to a restriction enzyme (HindIII). The DNA fragment
containing the 2.0 λ fragment was determined by comparing the electrophoresed cut DNA to known standards and also
observing that two highly conserved sequences were seen on the gel after electrophoresis. Through the use of ligation and
transformation, DNA cloning was accomplished. Tests were run that involved electrophoresis and digestion by HindIII in
order to fingerprint the DNA. The clone was identified as the desired sample through the use of transformation to identify
white colonies (those with the insert) and blue colonies (those lacking the insert). The DNA molecule desired was then
isolated through the use of a special resin that binds to DNA at high salt and not at low salt. The DNA is purified and the
cloned sample is obtained. To verify that the cloned sample contains the correct ligating insert, digestion of the DNA was
performed with HindIII and electrophoresed. The samples were found to contain the 2.0 kbp lambda fragment verifying that
the sample was indeed cloned by recombinant DNA technology.
Key words: DNA Technology, Cloning, Genetics, Biotechnology.
Introduction
Over the course of this lab experiment, recombinant
DNA technology was used to clone specific DNA
fragments. There were several steps to this procedure that
utilized various forms of technology and information.
The steps performed involved analysis of the anatomy
and evolution of the genome, genetic fingerprinting of
an unknown sample, the recombinant DNA technology
which can be split into ligation, fingerprinting, miniprep,
and digestion (Leisner and Shemshedini, 2009).
To fully understand the results and significance of
cloning DNA through the use of recombinant DNA
technology, a basic understanding of DNA must be
obtained. DNA is the genetic material of all living
organisms and some viruses. DNA is composed of sugardeoxyribose, phosphates, and nitrogenous bases. DNA
is double-stranded, and the sequence of bases found in
DNA is a way of storing genetic information. DNA is
either replicated or transcribed into RNA, which is then
translated into proteins that determine what happens
within cells (Leisner and Shemshedini, 2009). The
School of Doctoral Studies (European Union) Journal - 2010
2010
Cloning DNA through the use of Recombinant DNA Technology
genome in an organism is its total complement of DNA.
The size of DNA is quantified by giving them in terms
of base pairs or in kilobase pairs for large genomes.
There are two major groups of organisms: Eukaryotes
and Prokaryotes. These classes are based on genome
type. DNA is also found in non-cellular entities such
as plasmids, viruses, and cellular organelles. Genome
size is directly related to complexity of a life form. The
larger the genome size is, the more complex the life form
(Leisner and Shemshedini, 2009).
Plasmids are DNA molecules that are circular in shape
and found in most species of bacteria that exist apart
from the bacterial chromosome. These molecules encode
a small number of genes. Many of the genes that allow
bacteria to be resistant to antibiotics are found on plasmids.
Viruses are non-cellular organisms and composed of
protein and nucleic acid. Cellular organelles contain
small circular DNA molecules that encode a few genes.
The method used to compare various DNA molecules
is to measure the distance migrated from the point of
insertion in an electrophoresed gel and to plot it against
the log of the molecular weight. Gel electrophoresis is
the placement of a gel containing samples of DNA, in
this case, into SDS-agarose (a detergent) for denaturing
and an electrical charge is put through the medium. The
DNA have been stained prior to insertion so that the
bands will be visible under ultraviolet light (Leisner
and Shemshedini, 2009). The DNA molecules carry a
negative charge and move toward the positive electrode.
The distance migrated from the point of insertion can be
measured and compared to a set known of standards that
form a calibration curve in order to obtain the molecular
weight of the DNA (Leisner and Shemshedini, 2009).
The main difference between electrophoresis of DNA
from proteins is that the DNA is digested with restriction
enzymes. Restriction enzymes are proteins that cleave
DNA at specific locations. A restriction enzyme will
cut the DNA at a specific sequence that it recognizes.
The cleavage can be blunt or sticky ended. When two
sticky ends glue together with another enzyme called
DNA ligase specific DNA fragment cloning is possible
(Leisner and Shemshedini, 2009).
A genetic fingerprint is the specific patter of restriction
enzyme-generated fragments. An example of such a
genetic fingerprint comes in the case of the comparison
between Herpesviridae and Iridoviridae families (Leisner
and Shemshedini, 2009). Herpesviridae’s specific pattern
is that when cut by restriction enzyme HindIII, two highly
129
conserved DNA sequences are found. In Iridoviridae
only a single highly conserved DNA sequence is found
when cleaved with HindIII (Leisner and Shemshedini,
2009).
Ligation and transformation of DNA into bacteria
is the initial step in molecular cloning. An insert (the
specific DNA molecule to be cloned) is produced with
proper ends by cleavage with a restriction enzyme.
The insert is then joined to the vector (another DNA
molecule) by DNA ligase (Kornberg 2005). The vectorinser molecule is then propagated with bacteria through
a process known as transformation. The vector in this
case is a plasmid, pUC18, which is propagated with host
bacteria (E. coli) (Kornberg 2005).
The way of distinguishing between the bacteria that
have taken up the vector with insert and those that have
not depends on β-galactosidase. If bacteria have taken up
the vector with insert then they will be able to grow on
plates containing ampicillin, an antibiotic (Leisner and
Shemshedini, 2009). If it does not have the vector with
insert, then the bacteria will not propagate on the plate
containing ampicillin. Another means of determining
which bacteria haven taken up the insert is by determining
the color of the bacteria when exposed to X-GAL. The
β-galactosidase gene splits the compound X-GAL into
X + GAL. When X is freed, a blue color is seen. A blue
colony demonstrates that the insert was not taken up, and
a white colony establishes that the bacteria contain the
insert (Reddi, 2000).
Minipreparations are performed on the ligated,
digested, and inoculated bacteria. A miniprep is the
isolation of DNA from small bacterial cultures. The
process for the miniprep is to pick and grow specific
bacterical colonies from a transformation plate containing
both blue and white colonies. From the liquid culture,
the bacteria will be concentrated, broken open, and the
plasmid DNA will be purified (Leisner and Shemshedini,
2009). Purification is based on the idea that DNA, when
present at high salt, binds to a special resin and does not
bind to the same resin at low salt. A cell lysate is then
run over the column to purify plasmid DNA. When the
plasmid binds to the column, the rest of the bacterial cell
materials will be washed away (Reddi, 2000). The DNA
is the released from the column giving a pure preparation
of plasmid DNA (Leisner and Shemshedini, 2009). The
DNA is then digested with HindIII to determine which
1 DNA fragments have been cloned. The overarching
hypothesis for this entire process is that by using
Rotenhoffen E. - Cloning DNA through the use of Recombinant DNA Technology
School of Doctoral Studies (European Union) Journal
130
recombinant DNA technology, one can clone specific
DNA fragments (Leisner and Shemshedini, 2009).
Materials and Methods
The materials and methods can be found verbatim
within the Fundamentals of Life Science I Lab Honors
manual by Dr. Scott Leisner and Dr. Lirim Shemshedini,
except where noted in the following. In the lab for
DNA fingerprinting, the standards did not come out in
a readable fashion so the standards readings obtained
in the lab for analysis of the anatomy and evolution of
the genome was used. For the white colonies used in
inoculation and later for isolation and digestion, a liquid
colony with already prepared bacteria with insert was
used because the colonies on Transfomation 4 were
satellite colonies and unusable.
Results
The results that will soon be presented are used
to either refute or support the hypothesis that using
recombinant DNA technology allows for the cloning of
specific DNA fragments. Several tests were performed in
order to test this hypothesis. The anatomy and evolution
of the genome was under study. Genetic fingerprinting
2010
was performed. Recombinant DNA procedures were
used to attempt to clone specific DNA fragments, and the
results of all these tests follow. 3
The anatomy and evolution of DNA was under
examination. The purpose of this lab was to examine
the complexity of different genomes, determine if all
of the DNA within different cells of an organism is
the same, and determine the size of DNA molecules.
The complexity of different genomes was determined
based on the principle that the larger the genome size,
the more complex the life form. The size of the DNA
molecules is determined by electrophoresing the samples
and comparing the distances they travel from the point
of insertion to known standards, in this case the standard
is the restriction enzyme map for lambda bacteriophage
DNA, and plotting their log base 10 values on a graph to
determine molecular weights (Table 1). In Graph 2, the
lambda bacteriophage DNA standards are graphed for
comparison with the plasmid DNA and the calf DNA.
It was determined that the DNA within different cells of
an organism are the same by comparing the distances the
DNA traveled and the amount of bands in the calf thymus
DNA and the calf kidney DNA, if the distances traveled
are the same, and the amount of bands is equal then the
different cells of an organism are the same (Table 1 and
Graph 1).
Figure 1. Photograph of UV Transilluminated Electrophoresed Gel of Plasmid DNA, Lambda bacteriophage DNA, Calf Thymus
DNA, and Calf Kidney DNA
P λ
T K
23.1 kb
9.4 kb
6.6 kb
1.44 kb
4.4 kb
2.77 kb
2.3 kb
1.44 kb
P = Plasmid DNA
2.0 kb
λ = Lambda Bacteriophage DNA
T = Calf Thymus DNA
0.56 kb
K = Calf Kidney DNA
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Cloning DNA through the use of Recombinant DNA Technology
2010
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Table 1. Distance Migrated and Size of DNA Molecules for Standards, Calf DNA, and Plasmid DNA
Distance Migrated
by Standards (cm)
DNA Fragment Length
Log Kilobase Pairs
Sizes of DNA
molecules in
Standards
3.7
1.36
23.1
4.5
0.97
9.4
5.0
0.819
6.6
Plasmid DNA
7.2
0.443
2.77
5.8
0.643
4.4
8.6
0.158
1.44
7.3
0.361
2.3
Calf thymus
DNA (satellite)
7.5
0.301
2.0
Calf kidney DNA
(satellite)
8.6
0.158
1.44
11.1
-0.252
0.56
Sample
Distance
Migrated
(cm)
DNA Fragment
Length Log
Kilobase Pairs
Sizes of DNA
Molecules
(kb)
Graph 1. Lambda Bacteriophage DNA Log Base 10 of Molecular Weight versus Distance Migrated
Genetic fingerprinting was performed using restriction
enzymes to distinguish different virus genomes. This
was accomplished by comparing an unknown virus
with known characteristics of being an enveloped
double-stranded, DNA-containing virus temporarily
named KDV to two viruses of known molecular weight
that match the unknown viruses characteristics. By
comparing the sizes of the KDV DNA fragments with
DNA standards of known sizes, the family that KDV
belongs to can be determined. This is accomplished by
electrophoresing the unknown sample, the standards,
and the two virus families (Figure 2) and graphing the
distance the samples traveled from the point of insertion
to determine the molecular weight of the KDV DNA
to determine its identity (Graph 2). By determining
the molecular weight of the unknown, the weight and
identity of the KDV DNA can be determined (Table 2).
Also in Table 2 is the log base 10 values of the Standard
DNA standard weights, but the method for obtaining
the molecular weight of the DNA was changed slightly.
The Standards distance traveled could not be measured,
because they traveled off the gel. This error was remedied
by comparing the distance traveled by the two known
viruses (Herpesviridae and Iridoviridae) and unknown
virus (KDV) to the previously run standards that had
been used in the lab studying the evolution of DNA.
The two known viruses belong to the Herpesviridae
and Iridoviridae families. The main difference between
Rotenhoffen E. - Cloning DNA through the use of Recombinant DNA Technology
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132
2010
the Herpesviridae family of viruses and the Iridoviridae
family is that the Herpesviridae family of viruses
contains two highly conserved DNA sequences that are
cut by the Restriction enzyme HindIII, and when the
same process is performed on Iridoviridae one conserved
HindIII site is seen. Graph 2 displays the molecular
weights and the distance traveled by the standards. The
virus that will be taken from the Herpesviridae family is
the Herpes Simplex Hemorrhagic Virus (HSHV), and the
virus from the Iridoviridae virus family is the Chicken
Hemorrhagic Anemia Virus (CHAV). Table 2 contains
all the molecular weights of the unknown, known, and
standard DNA fragments with the log base 10 values
and the distance traveled from the point of insertion for
the unknown and known DNA fragments. No bands of
unusual size are seen in Figure 2, showing that the test
went just as expected except for the standards going off
the gel.
Figure 2. Picture of Electrophoresed Gel Containing
Standards, HSHV Cut and Uncut, CHAV Cut and Uncut, and
KDV Cut and Uncut
Table 2. Molecular Weights of Standards, CHAV, KDV, and
HSHV and the Distance Traveled in Electrophoresed Gel by
CHAV, KDV, and HSHV
Distance
Migrated
(cm)
DNA Fragment
Length Log Kilobase
Pairs
Molecular
Weight
(kbp)
Cut HSHV 1
4.5
0.301
2
Cut HSHV 2
4.3
0.431
2.7
Cut CHAV
3.6
0.672
4.7
Uncut HSHV 1
3.1
0.877
7.5
Uncut HSHV 2
3.9
0.561
3.6
Uncut CHAV 1
3.3
0.798
6.3
Uncut CHAV 2
4.2
0.443
2.8
Uncut KDV 1
3.1
0.877
7.5
Uncut KDV 2
3.9
0.561
3.6
Cut KDV 1
4.5
0.301
2
Cut KDV 2
4.3
0.431
2.7
Molecular Weights
of Standards (kbp)
Distance Migrated
(cm)
DNA Fragment Length
Log Kilobase Pairs
23.1
3.7
1.36
9.4
4.5
0.97
6.6
5.0
0.819
S = λ Bacteriophage DNA Standards
4.4
5.8
0.643
HU = HSHV Uncut
2.3
7.3
0.361
2
7.5
0.301
0.56
11.1
-0.252
S
HU HC KU
KC CU
CC
Sample
6.3 kbp
7.5 kbp
2.7 kbp
2.7 kbp
7.5 kbp
4.7 kbp
3.6 kbp
3.6 kbp
2.8 kbp
2.0 kbp
HC = HSHV Cut with HindIII
KU = KDV Uncut
2.0 kbp
KC = KDV Cut with HindIII
CU = CHAV Uncut
CC = CHAV Cut with HindIII
School of Doctoral Studies (European Union) Journal - 2010
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Cloning DNA through the use of Recombinant DNA Technology
133
Graph 2. The Standards from Graph 1 used to Compare Against to Discover the Molecular Weight of KDV Virus Fragments
The initial steps in the recombination of DNA
were to clone 2.0 kbp λ-DNA fragment generated by
HindIII digestion into the plasmid pUC18. This was
accomplished by producing large amounts of the specific
DNA molecule (pUC18) so that it may be manipulated.
The insert (pUC18) is cleaved with HindIII and ligated
back together in ligation. Ligation is the process of
joining the insert to another DNA molecule (the vector)
through DNA ligase. Once the insert was ligated to the
vector, it was introduced into host bacteria. The bacteria
then begin to propagate rapidly, which in turn leads to
the production of large amounts of the vector-insert
molecule. The bacteria in this lab were Escherichia
coli (E. coli). The process of producing large amounts
of vector-insert molecule through the introduction to
bacteria is called transformation. The bacteria that take
up the pUC18 with the insert will be white and have no
functional enzyme. The production of β-galactosidase
gene splits the compound into X + GAL instead of
X-GAL. The bacteria colonies that express a nonfunctional β-galactosidase enzyme appear white on
plates containing X-GAL.
The purpose of the second part in the recombination of
DNA was to isolate plasmid DNA from E. coli harboring
pUC18 plasmids with λ-DNA fragment inserts and cut the
DNA with HindIII. Bacteria from Transformation 4, both
blue and white colonies were to be taken for inoculation.
The white colonies could not be obtained because they are
satellite colonies that grow from enzyme excretion. The
samples were obtained from already prepared liquid forms
of the white colonies. The inoculation was performed
by streaking a blue colony from Transformation 4 and
five white colonies from separate liquid forms onto a LB
AMP X-GAL plate. These samples were also placed in
a liquid culture of LB AMP medium. Concentrating the
bacteria then breaking them open for purification was the
first step to isolating the plasmid DNA from E. coli that
had the pUC18 plasmids with λ-DNA fragment inserts.
The DNA was purified based on the idea that DNA,
when present at high salt, binds to Wizard Minipreps
DNA Purification Resin. A cell lysate is then run over
the column to purify the sample. The plasmid DNA is
now bound to the column and the rest of the cell will
be washed away. The DNA is then released from the
column giving a high purity preparation of plasmid
DNA. The samples are digested with HindIII in order
to determine which 1 DNA fragments have been cloned.
The two transformation 2 plates were compared, and the
T2 with the LB AMP X-GAL had no colonies (although,
it should have had blue colonies). The T2 without
AMP had smeared bacteria because no antibiotics were
present. The number of colonies found on the well plates
was recorded as well as there color (Table 3). The color
of the streaked columns from Transformation 4 is also
recorded (Table 4).
Rotenhoffen E. - Cloning DNA through the use of Recombinant DNA Technology
School of Doctoral Studies (European Union) Journal
134
Table 3. The Characteristics of the E. coli Strands on
the Plates
T1
T2
T3
T4
0
0
0
12
N/A
N/A
N/A
Number of
colonies
Color of
colonies
T2 (LB)
2010
Figure 3. Picture of Electrophoresed Gel containing Known
Standards, Uncut DNA from Blue Colony, Cut with HinDIII
DNA from Blue Colony, and 5 Cut DNA with HindIII from
White Colonies
Smeared
Blue and
White Satellite
Not
Distinguishable
Table 4. The Characteristics of the Streaked Samples from the
Tranformation 4 Plate
Streaked
Column Number
Color
1
Blue
2
3
4
5
6
White
White
White
White
White
The final procedure for recombinant DNA was to
identify plasmids that have the 2.0 kbp λ-DNA fragments.
This was accomplished by running the digested DNA
HindIII on a gel to separate the DNA fragments (Figure 3).
The colonies were compared to blue uncut and cut DNA
and known standards (Figure 3). The molecular weight
of the fragments was determined, and by discovering
the molecular weights of the fragments and comparing
them to the known weight (2.7 kb) for the pUC18. The
image of the electrophoresed gel is seen in Figure 3. The
distance traveled by each of the DNA fragments was
recorded and placed in Table 4, the standards were then
graphed with their molecular weights against how far
they migrated from the point of insertion so as to obtain
the molecular weights of the DNA fragments in the blue
and white colonies (Table 5 and Graph 3). No bands of
unusual size were seen in the white colony samples,
but the blue colony sample did contain a band that was
found to contain a DNA fragment of 2.5 kb. After all
the samples have been digested and electrophoresed,
the type of E. coli strains are determined based on their
characteristic of whether or not they contained the insertvector molecule or were simply E. coli with the vector
or had a different connecting sequence length from 2.0
kb (Table 6). From the table it was obtained that all the
original colonies (pJB2-pJB3) contain the 2.0 kb λ DNA
fragment. No bands of unusual size were seen on the
electrophoresed gel (Figure 3).
S = Standard DNA
BU = Blue Colony Uncut
BC= Blue Colony Cut by HindIII
W1 = White Colony Cut by HindIII
W2 = Different White Colony Cut by HindIII
W3 = Different White Colony Cut by HindIII
W4 = Different White Colony Cut by HindIII
W5 = Different White Colony Cut by HindIII
Table 5. The Molecular Weights and Distance Traveled by the
Standards, the Blue Colony both Cut and Uncut, and the Five
Cut White Colonies
Distance
Migrated (cm)
DNA Fragment
Length Log
Kilobase Pairs
Sizes of DNA
molecules (kb)
3.1
0.699
5
3.3
0.602
4
3.7
0.477
3
3.9
0.398
2.5
4.3
0.301
2
4.8
0.176
1.5
5.8
0
1
6.4
-0.125
0.75
7.2
-0.301
0.5
8.2
-0.602
0.25
School of Doctoral Studies (European Union) Journal - 2010
Sample
Standards
Cloning DNA through the use of Recombinant DNA Technology
2010
Distance
Migrated (cm)
DNA Fragment
Length Log
Kilobase Pairs
Sizes of DNA
molecules (kb)
Sample
N/A
N/A
N/A
Blue Uncut
4.1
0.392
2.7
Blue Cut
4
0.416
2.7
White 1
4.4
0.321
2.0
White 1
4
0.416
2.7
White 2
4.4
0.321
2.0
White 2
4
0.416
2.7
White 3
4.4
0.321
2.0
White 3
4
0.321
2.7
White 4
4.4
0.321
2.0
White 4
4
0.416
2.7
White 5
4.4
0.321
2.0
White 5
135
Graph 3. The DNA Standards Molecular Weights Versus the
Distance Migrated
Table 6. The Characteristics and Plasmid Names of the Different E. coli Strains Created
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School of Doctoral Studies (European Union) Journal
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Calculations
For each lab standard graph, the log base 10 was taken
of the molecular weight and plotted against the distance
migrated from the point of insertion.
Example (From Table 1)
log (molecular weight) = log base value
log (23.1) = 1.364
To determine the molecular weight of the unknown
samples being tested in each case, the distance migrated
values were plugged into the trend line equation and
that value was then used as a power of ten to obtain the
molecular weight. The example is taken from the plasmid
DNA in Table 1.
Example (From Table 1)
x = distance migrated (cm) y = log base 10 value of
molecular weight
y = -0.204x + 1.912
= -.204(7.2) + 1.912
= 0.443
To find the molecular weight the log base 10 value is
taken as a power of ten.
Molecular weight = 10(log base 10 value)
= 100.443
= 2.77 kbp
Discussion
The hypothesis for the entire experiment that
was performed was that by using recombinant DNA
technology, one can clone specific DNA fragments. The
results were used to determine whether or not this claim
is supported.
In testing the anatomy and evolution of the genome,
the larger the genome size the more bands were found
when the DNA was electrophoresed. In the example found
2010
in Figure 1 and Table 1, the plasmid DNA was found to
have one band signifying that it had a small genome, and,
therefore, was low in complexity compared to the calf
thymus and kidney DNA. The calf thymus and kidney
DNA are shown to have a high complexity based on how
the electrophoresed gel containing the DNA was a giant
streak for each sample. The samples were also identical
when electrophoresed, the streaks were the same and the
satellite band that was visible in each sample was in the
same place for both. Table 1 showed that the satellite for
each was 1.44 kb in molecular weight. This would lead
to the conclusion that the DNA in the kidney of the calf
was the same as the DNA in the thymus. The satellite
and plasmid bands were of relative similarity to the maps
provided for comparison. The procedure ran smoothly
and the weights and distances migrated of all the samples
were easily determined.
Genetic fingerprinting was also performed using an unknown
virus labeled KDV and comparing it to a set of standards
and two known viruses (HSHV and CHAV). The sizes of
the HSHV bands seen in Figure 2 are as expected. The
cut HSHV DNA has two bands one weighing at 2.7 kb
and the other 2.0 kb, just as was described. KDV was
found to belong to the Herpesviridae family of virus
as seen in Table 2. The results showed that the bands
and molecular weights of the DNA fragments for KDV
matched perfectly with that of HSHV, which comes from
the Herpesviridae family. Although the Standards did
not come out as expected, this was remedied by using
previously obtained standards. Everything else went as
expected and the results supported the hypothesis.
The next section of the procedure in DNA cloning
revolves around recombinant DNA technology. This
process works because of the development of methods for
propagating large amounts of a specific DNA molecule.
This process involves the production of a specific DNA
molecule (the insert) in large amount so that it may be
manipulated. The insert is the produced with proper ends
for the cloning by cleaving with a restriction enzyme.
This insert molecule is then jointed to the vector (another
DNA molecule) by DNA ligase, which utilizes ATP.
Transformation is the performed, which is the introduction
of the vector-insert molecule into bacteria. The bacteria
are allowed to propagate and large amounts of the vectorinsert molecule are created. The procedure did not go
exactly as planned. The Transformation 2 should have
had blue colonies but none formed, signaling an error in
School of Doctoral Studies (European Union) Journal - 2010
2010
Cloning DNA through the use of Recombinant DNA Technology
procedure. Transformation 4 also had a problem in that
the white colonies that formed could not be used because
they were satellite colonies.
The second part of the recombinant DNA procedure
involved the isolation and digestion of the DNA in a
process known as a miniprep. The colonies stayed white
after patching on the new plate indicating that a sample
of the desired DNA with insert has been created. When
Transformation 2 was compared with the Transformation
2 that was placed on the plate that had only LB, it
allowed for the visualization of the effectiveness of
the antibiotic. Even though blue colonies should have
formed on the Transformation 2 with LB AMP X-GAL,
this did not deter the fact that the antibiotic did mutate
the bacteria from its original state. The Transformation
2 with just LB was seen as being a smeared plate
indicating the existence of large unquantifiable bacteria
existence. There were two problems that occurred during
the miniprep. The problem being the nonexistence of
blue colonies on Transformation 2 and the existence
of unusable satellite colonies on Transformation 4.
This problem can be attributed to a procedure error in
which the bacteria were probably incorrectly placed onto
the plate. The final electrophoresed gel gave results
137
showing that five bacterial colonies harboring the 2.0 λ
DNA fragment. The blue uncut colony did not show up
as having a band, and the cut blue colony only showed
a 2.7 kb band (Figure 3). There were no unusual bands,
leading to the conclusion that the experiment ran as
expected. The names of the plasmids are as follows:
pJB1 (pUC18), pJB2, pJB2, pJB3, pJB4, pJB5, pJB6.
PJB2-pJB6 came from the white colonies.
The hypothesis that using recombinant DNA
technology one can clone specific protein was supported
by the fact that 5 colonies were created that contained the
specific 2.0 λ DNA fragment, signaling that the bacteria
contained the desired insert-vector molecule.
References
Kornberg, A., & Baker, T. (2005). DNA Replication.
University Science Books.
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