Practical Report: DNA Manipulation and Construction of pBSK II-FtsZ Vector

This practical report discusses the process of DNA manipulation and the construction of pBSK II-FtsZ vector. It includes steps such as plasmid isolation, restriction endonuclease digestion, gel electrophoresis, and ligation. The report also provides details on the methods used and the results obtained.

Practical Report
PRACTICAL ONE: DNA MANIPULATION
Introduction
The practical session entailed the isolation of pBlue Script KS II(+) plasmid from an overnight E.coli
culture. Subsequently, restriction endonucleases BamHI and HindIII was used to digest donor plasmid
pProEX and the recipient isolated plasmid pBKSII. The digestion of the donor plasmid was aimed at
isolating the Pc-fitsZ that would then be inserted into the pBKSII plasmid. Restriction endonucleases like
BamHI and HindIII are bacterial enzymes that have the capability to cleave base pairs within double-
stranded DNA. BamHI and HindIII are highly selective type 2 restriction endonucleases that act as useful
tools in molecular biology research where manipulation of genetic material is key for research. Inside the
bacterial hosts of the restriction enzymes are used in destruction of any foreign DNA that invades their
system like the bacteriophages. These restriction enzymes possess different properties that define their
activity like temperature and ionic strength of the buffers used during practical sessions.
Plasmids are extrachromosal genetic components of bacterial cells. They are of different sizes that could
range from 1Kbp to 200kbp. The plasmids contain genes that enable the synthesis of different enzymes
providing advantages to the bacterial host like antibiotic resistance capabilities. In research, plasmids are
used as vectors whereby the genes of interest are inserted through ligation processes to generate a
recombinant molecule. Recombinant plasmids are plasmids containing genes from more than one source.
For instance, the host cell’s DNA and the DNA of interest. The recombinant plasmid is then introduced
into the preferred host cell through a transformation process to allow for replication of the DNA of
interest. There are certain traits that laboratory plasmids must contain which include origin or replication,
multiple cloning site and a resistance gene that would allow for the identification. These characteristics
work synergistically to enable successful DNA manipulation in molecular techniques like genetic
engineering.
The steps followed during the isolation of the plasmid entails culturing of the host cells like E.coli for the
case of this practical. Further, upon the growth of the cultured host cells, they are harvested and lysed to
release various cell components that include both chromosomal and plasmid DNA. Subsequently, there is
precipitation of DNA to isolate other cell protein components from the required genetic material. The
plasmid DNA isolation from a combination of the chromosomal DNA takes advantage of the longer
length of the chromosomal DNA relative to the small size of Plasmid DNA. During the isolation process,
the complex chromosomal DNA interact with the proteins elements forming a large aggregate that
precipitates. On the other hand, the small sized plasmids renature quickly and remains in the solution.
Upon the removal of the precipitate, the plasmid in the solution gets precipitated using ethanol for use as
a vector. During this practical it was expected that pBKSII would be isolated from E.coli. Further, it was
expected that pBKSII would be digested with BamHI and HindIII to excise the lacZ sequence from the
multi-cloning site. On the other hand, it was also expected that pProEX would be digested by HindIII and
BamHI.

Methods
Small Scale plasmid isolation
The 3 mL Luria Broth was inoculated with colony bacteria that contain the plasmid of interest pBKSII
and ampicillin. The culture was allowed to grow at 37°C overnight. 1 mL of the resulting culture was
transferred into the centrifuge. The supernatant from the centrifuge was discarded into the waste liquid
contained and the remaining pellet used for subsequent steps. The pellets are then resuspended in 100mL
of the resuspension buffer (GTE) that contains 50mM glucose, 25mM Tris-HCl with pH of 8.0,and
10mM EDTA with a pH of 8.0. The resulting mixture was mixed through vortexing to break any cell
clumps. 200 mL of freshly lysis buffer containing 0.2N NaOH and 1% Sodium Dodecyl sulfate (SDS)
was then added to the resuspended cells and then shaken followed by incubation. After five minutes 150
mL of the neutralization buffer containing 3M potassium acetate was added followed by incubation for
five minutes. The solution got centrifuged for 10 minutes at top speed and 400uL of the supernatant
transferred into a new tube labeled as pBKSII. The resulting DNA is then washed by three times to
remove any impurities using ethanol. The first washing was undertaken using 100% ethanol and the
centrifuged. The resulting DNA was then washed using 1mL of 70% ethanol and then finally with 0.5mL
of 70% ethanol followed by centrifugation. The resulting pellet is dried well to remove any residual
ethanol from the tube. The pellet was then resuspended using 40μL of of 10mM Tris-HCl containing
RNase A noted as Elution Buffer EB. After incubation the resulting solution is divided into two parts
labeled for southern Blot and the other for digestion using the restriction enzyme and then stored at -
20°C.
Restriction Endonuclease digestion of the donor pProEX plasmid and recipient plasmid pBKSII
The preparation of the restriction mixture was done separately for both the pProEX and pBKSII plasmids.
For the pProEX plasmid pProEX +FtsZ digestion that got labeled as tube 4 entailed the addition of 6μL
of deionised water, 3μL of 10X buffer 2, 3μL of BamHI, and , 3μL of HindIII and finally 15μL of
pProEX +FtsZ DNA. On the other hand, the tube labeled “Tube 3” for pBKSII plasmid was added into
11 μL of deionised water, 3 μL of 10x Buffer 2 solution, 3 μL of BamHI, 3 μL of HindIII and then finally
10 μL of pBKSII brought from the miniprep practical session. Notably, new pipette tips were used for
every step and the final volume of each mixture was 30 μL in volume. The restriction endonuclease
digestion tubes then got incubated at 37° C for duration of one hour in heat block. The underlying
digestion was then stopped by incubating the tubes at 65° C for duration of 10 minutes.
Results
Exercise 1A
The practical was conducted well and a solution of the required recipient plasmid DNA (pBKSII)
appearing as a whitish pellet upon isolation and washing, labeled and stored for the subsequent practical
sessions. Part of the PBS KS II isolated was used for restriction endonuclease digestion and the other part
stored for use during the Southern Blot procedure later.

Exercise 1B
The outcome of the exercise was positive, the pBKSII and pProEX +FtsZ DNA got digested using
restriction endonucleases BamHI and HindIII. The tubes containing the digests labeled as Tube 3 and
Tube 4 got stored for subsequent practical.
Discussion
The isolation of plasmid requires its growth in a bacterial cell culture. During the practical, Lubia broth
was used for the inoculation of the bacteria. The use of ampicillin in the culture was to help in the
selection of only bacteria that are resistant to ampicillin to allow for selection of transformed bacterial
cells later. Lack of the antibiotic can lead to eventual loss of the plasmid containing the genetic marker.
Centrifugation allows the contents of the culture to mix uniformly and also even distribution of contents
like nutrients and antibiotics. The bacteria were then pelleted and resuspended in the resuspension buffer
prepared. The resuspension process should be done to completely remove any cell clumps so that all cells
are exposed to the lysis buffer. The basic Tris buffer for resuspension helps in the denaturation of the
DNA accompanied by EDTA that acts to bind the divalent cations to destabilize the membrane and
destroy DNases. RNases are added to destroy any RNA released from the cell.
The detergent SDS used in the lysis step solubilizes the phospholipids and proteins making up the cell
membrane and hence releasing the cell contents. Further, the use of NaOH in high concentration
denatures the plasmid and genomic DNA as well as other intracellular proteins. This leads to linearization
of the DNA and breakage of the duplex strand. Given the small size of the plasmid DNA that is circular,
their denatured strands remain close together increasing chances of renaturation.
The neutralization buffer acts to neutralize the strong basic conditions. Addition of potassium acetate into
the solution facilitates the formation of a high salt concentration and hence development of a white
precipitate containing SDS, lipids and proteins. Neutralization also enables renaturation of plasmid DNA
to occur. Large chromosomal DNA remains in the precipitate while the small sized plasmid DNA remains
suspended in the solution. Centrifugation allows for the removal of the precipitate and the genomic DNA.
Consequently, the soluble extrachromosomal plasmid DNA is then purified by the use of isopropanol.
Isopropanol precipitation is performed at room temperature to minimize the probability of salt co-
precipitation that would adversely affect downstream processes.

PRACTICAL TWO: CONSTRUCTION OF pBSK II-FtsZ VECTOR
Introduction
The products of a DNA restriction endonuclease digestion are genetic fragments of different sizes.
However, for the purposes of vector development there is need to select the most appropriate sizes for the
insertion of the selected gene into the vector. Hence, there are certain steps that are required for the
development of a vector in molecular biology. These include gel electrophoresis, purification of the
DNA bands, and ligation of the DNA to a selected target plasmid DNA.
Gel electrophoresis is a molecular separation technique that enables separation of molecules based on
their relative sizes. The molecules like pieces of DNA that have been digested by restriction
endonucleases are loaded into wells and then an electric field is applied. The fragments of DNA then get
separated towards the positive end of the electric field. This is because DNA molecules are negatively
charged. Thus, the separation speed and distance will only depend on their relative sizes, hence making
small particles to migrate faster covering the longest distance on the gel. The movement of the DNA
across the gel depends on factors that include molecular size of the DNA molecule, concentration of the
agarose used in gel preparation, conformation of the DNA fragment used and the amount of current
applied during the gel electrophoresis process.
Visualization of the DNA being analysed is achieved by the use of ethydium bromide that enables
visualization of the DNA bands by illuminating them with ultra violet light. Care should be taken while
handling ethydium bromide because it is carcinogenic and can cause adverse health effects to researchers.
Gel electrophoresis step also utilizes a ladder containing DNA fragments of known sizes that would help
in the estimation and selection of the right DNA fragments that can be used in the vector development
process. The selected fragments are excised and then purified using the spin-column method. These
columns contain high-affinity silica filters that attract DNA leaving other contaminants to be eluted.
Finally, the DNA of interest is eluted using appropriate buffer solution for further analysis.
A recombinant DNA is made by litigation of two DNA fragments. Recombinat DNA technology is often
used in the introduction of foreign genetic material into a host cell DNA. The introduced genetic material
often confers certain characteristics that would beneficial to the recipient cells. These may include
drought resistant abilities, antibiotic resistance, and pest resistance among others. The process proceeds
through a litigation process that requires the presence of a DNA ligase enzyme. The choice of the DNA
ligase will depend on the objectives of the research and the decisions of the researchers that may include
the types of the DNA ends to be ligated whether sticky or blunt, stability of the hydrogen bond structure
of the DNA fragments, temperature, the concentration of the DNA fragments, and the length of the sticky
ends to be ligated. The ligase enables the binding of the DNA fragments with either blunt or sticky ends
through catalysis of the formation of a phosphodiester bond between the 3’OH and 5’phosphate ends of
adjacent termini in double stranded DNA. This reaction would often require the presence of magnesium
ions and ATP as major cofactors. During this practical it was expected that the digested DNA fragments
would be of the expected molecular weight as measured against the standard used.

Methods
Exercise 2A: Gel Electrophoresis
The practical proceeded through gel preparation that was done by the instructor. The gel prepared was 1%
agarose in 1% TAE boiled in a microwave and then allowed to cool to about 50°C.Ethydium bromide was
then added, then the gel poured into the casting tray and combs inserted. Subsequently, the sample
preparation was done. 6 μL of 6X gel-loading buffer was added into the “Tube 3” that contains pBKSII
DNA fragments that had previously been digested with BamHI and HindIII and then mixed. Similarly, 6
μL of 6X gel-loading buffer was added to “Tube 4” that contains DNA fragments of pProEX + FtsZ that
had been digested by HindIII and BamHI and the content shaken to mix well.
The gel was the placed into the tank followed by addition of 1X TAE buffer covering the gel by about 0.5
cm. The instructor added 5 μL of the molecular weight marker into the first and the last wells of the gel.
One well was left empty after each sample addition to avoid contamination. The samples got loaded
carefully by drawing 20 μL of pBKSII digest that had been cut by BamHI and HindIII, any residues from
the tip of the gel got wiped using lint-free tissue then the sample is loaded slowly into the well. This
process was repeated for the pProEX +FtsZ digest. The instructor then loads 5 μL of controls containing
undigested PBKS II and pProEX DNA mixed with 2 μL of gel loading buffer each. Subsequently, the
cover of the electrophoresis unit was placed and then 160V of electric current applied for 40 minutes.
Upon completion of the gel electrophoresis, power was switched off and the leads removed. The gel tray
was then carefully removed from the tank and transferred onto a plastic container. The instructor then
takes a picture of the gel before excision process ensues.
Exercise 2B: Excision and Purification of DNA Bands
This procedure was done in a group. Two sterile 2mL screw cap tubes are weighed; the weight is
recorded on the sides of the tube. One tube was labeled plasmid and the other as insert. The gel was then
placed onto the UV light and then bands were cut accordingly minimizing the size of the gels without
disrupting the fragment. The sliced gels then got transferred into the pre-weighed and labeled screw cap
tubes then weighed again. The mass of the gel size in the tubes then got calculated. 3 μL of QG buffer
containing 5.5M guanidine thiocyanate, 20mM Tris-HCl at a pH of 6.6 and 25 °C was added to each mg
of gel. The mixture was then placed under incubation at 50°C for 10 minutes and then inverted several
times to ensure the gel was completely dissolved. Upon complete dissolution of the gel, equal volume of
isopropanol to the amount of gel used is added into the mixture and then mixed well. Spin columns then
got placed in the 2mL collection tubes that are appropriately labeled for each group with sample ID and
initials. About 700μL of samples were then added to the spin columns and centrifuged for 10000 x g for 1
minute. The filtrate got discarded into collection waste and the spin columns returned to the same
collection tubes. The collection tubes were then emptied and 700μL of wash buffer (PE) containing
20mM of NaCl added to the spin columns and spun for 1 min at 10000 x g. The filtrate got discarded and
then columns spun for additional one minute to remove any residual buffer PE that could have remained.
Further, spin columns got placed into clean sterile and well labeled microfuge tubes whereby “Tube 3”
labeled plasmid had the linear PBKSII for southern blot while “Tube 4” labeled insert contained the
purified insert for southern blot. 30μL of water is then added to the centre of each spin column and
incubated for one minute at room temperature, centrifuged followed by DNA elution. The purified DNA
was then stored at -20°C awaiting subsequent practical activities.

Exercise 2C: Ligation of DNA
During this activity 4 μL of the linear pBKSII labeled “Tube 3” from the purification step was added to a
new tube together with 12 μL of Pc-FtsZ labeled Insert “Tube 4”, 2 μL of T4 DNA ligase and 2 μL of
10X ligation buffer. Subsequently, the tube got labeled as “Tube 5- Ligation” with group identifiers and
date on it. The contents then got mixed and centrifuged briefly then incubated at 4°C overnight. The tube
then got stored at -20°C waiting next practical session.
Results
Figure 1: Gel Electrophoresis Result.

According to the gel picture in diagram 1 above, the undigested control of pBKS II shows only one band.
The plamid was not digested and hence maintains its supercoiled conformation on the other hand, the
undigested pProEX plasmid shows two bands indicating the presence of both supercoiled and circular
conformations. The supercoiled conformation moves faster in the gel than the circular conformation. The
top most band is the supercoiled conformation for both plamids. The gel also indicates that pBKS II
double digested formed a single band while there was no observable band for digested pProEX.
Discussion
Gel electrophoresis is the separation of DNA fragments based on their relative sizes on an electric field.
The DNA fragments are negatively charged due to their phosphate group and hence are attar ted to the
anode. The concentration of the agarose gel chosen is dictated by the size of DNA fragments to be
separated. The agarose gel used is a matrix that is cross-linked with hydrogen bonds when cooled. When
the concentration of agarose is high smaller pores will result that may not efficiently separate the DNA
fragments well. The distances travelled by the DNA fragments was different based on relative sizes and
electrophoretic mobility, and conformations. The differential migrations enabled the prediction of their
molecular weights. The amount of glycerol used in the gel should be checked because it is much denser
than water and hence would weigh down the DNA fragments preventing their movement leading to bias
in the molecular weight estimations. The large DNA fragments would experience high frictional drag and
greater immobility within the matrix of the gel relative to small sized DNA fragments. Gels allow for the
separation and identification of DNA on the basis of their charge migration (Lee et al., 2012). Addition of
the gel buffer is made during sample preparation to increase the density of the plasmid DNA that should
be separated to ensure they sink into the wells.
Visualization of the separated DNA fragments in the agarose gels require the use of particular dyes. The
most widespread dye used for the visualization of Agarose gels is Ethydium bromide. It should be noted
that Ethydium bromide is mutagenic and toxic hence handled using nitrile gloves to avoid exposure to the
toxic chemical. Ethydium bromide can be added either before the electrophoresis or after the
electrophoresis process. However, addition of the Ethydium Bromide prior to the electrophoresis has
some advantages that include enabling of a faster and more convenient procedure, and allows monitoring

of the DNA fragments as they move through the gel. Some of the demerits to addition of ethydium
bromide prior to electrophoresis include the use of more ethydium bromide, mandatory decontamination
of the gel tanks and combs as well as reducing the electrophoretic mobility of linear DNA by about 15%.
On the other hand, the use of ethydium bromide after electrophoresis requires the use of less ethydium
bromide, requires no decontamination of the apparatus and no interference to the electrophoretic mobility
to the linear DNA. The demerit of ethydium bromide addition after electrophoresis reduces the speed of
the process by adding an extra step and also it does not allow for visualization of the migration process
(Tan and Yiap, 2009).
Ethydium bromide intercalates with the DNA sequences to aid in visualization. The complex fluoresces
when illuminated with UV light to allow the formed DNA bands to be seen. The image of the gel can then
be taken to be used later for analysis. The intensity of the UV light should be regulated if the DNA
fragments are to be used for downstream processes because strong UV light can fragment the DNA
further and damage them. Subsequently, the visualized DNA fragments are extracted from the gel through
excision by a sharp gel and then run through a spin column (Tan and Yiap, 2009).
The extraction of the DNA from the gel proceeds through three major steps that include binding, washing
and elution through a spin column. Addition of buffer QG introduces a chaotropic agent and a denaturant
that effectively disrupts the association between the DNA fragments and the gel as well as ethydium
bromide intercalations. The gels containing DNA are then heated to ensure all the gel has completely
dissolved to release DNA fragments. The pH of the buffer is maintained at acidity to ensure optimal
conditions are met for DNA binding onto the membrane. The silica membranes in the spin columns bind
the DNA fragments under conditions of high-ionic salt buffers. These conditions catalyze the formation
of hydrogen bonds between the silica and the DNA fragments (Tan and Yiap, 2009). During the
centrifugation process, the sample passes through the column allowing the binding to take place.
Upon the binding of the DNA to the silica, other contaminants such as proteins, contaminants and
nucleotides are removed using an alcohol-based wash which was isopropanol for this experiment. The
washes also ensure the salts are removed to allow elution of DNA. The DNA bound to the silica
membrane was then eluted using a low-ionic solution like sodium chloride which is then removed by
water for the final purification process. The low salt solution enables disruption of the hydrogen bonds
holding the DNA fragments to the silica of the spin column membranes. As such, the elution is done
under basic conditions at a pH of 8 to 9 that does not favor formation of ions (Gloeckner et al., 2009).
The final stage in the recombinant plasmid construction is the connection of the insert DNA gene in this
case FtsZ into the vector pBKS II that has already been digested. Ligation ensured covalent binding of the
sugar backbones of the DNA fragments that have been digested using similar restriction endonucleases
BamHI and HindIII. The endonucleases cleaved the DNA sequences at specific recognition sites creating
overhangs used during the ligation process. Linking of two sticky ends is much efficient relative to
linking of blunt ends. This is because the blunt ends require the use of linkers and at times polylinkers to
achieve their ligation. The reaction is catalyzed by T4 DNA ligase enzyme. The enzyme catalyses the
formation of phosphodiester bonds between the adjacent 3’OH and 5’ Phosphate group in the DNA
double strand (Kuwayama et al., 2002; McDaniel et al., 2010). After ligation, the recombinant plasmid
would then be used to transform competent bacterial cells.

PRACTICAL THREE: BACTERIAL TRANSFORMATION, SOUTHERN BLOTTING
Introduction
Bacterial transformation is the process through which competent bacterial cells are mixed with plasmid
DNA containing elements of foreign DNA segments to enable the plasmid to get into their cells. A
bacterial cells is referred to as competent when they have been treated with chemicals like calcium
chloride that allows water to get into the cells and make the swell (Sinha and Redfield, 2012). The
swelling interferes with the integrity of their membranes hence facilitating the entry of plasmids into their
cells. Upon transformation, the bacteria are then grown in culture containing antibiotics. When they
survive it means the transformation was successfully to confer resistance to the selected antibiotics. The
bacterial cells used for this practical are E.coli that are highly sensitive to ampicillin. As such, the
ampicillin sensitivity would allow for the selection of cells that have successfully been transformed in a
culture containing the ampicillin antibiotic. Further, the vector contains LacZ gene that enables blue/white
selection by coding for the enzyme β-galactosidase (Singh et al., 2010).
On the other hand, Southern blotting entails the transfer of DNA fragments from an agarose gel to a
membrane. It is meant to locate specific sequences of DNA within a complex mixture of strands. Notably,
the amount of DNA required to achieve Southern blotting depends on the size and specific activity of the
probe. Southern blotting is very elaborate and demanding entailing denaturation of the DNA while in the
gel, transfer of the denatured DNA onto a membrane, probing of the membrane with single stranded DNA
fragments, and visualization. Southern blotting is often used in the identification of DNA samples that
would be used for other molecular diagnostics (Loske et al., 2011; Opazo et al., 2012). It is also used to
confirm if a foreign DNA has been transferred successfully into a bacterial cell. Some of the applications
of Southern Blotting include the detection of genetic mutations, DNA fingerprinting, and gene cloning
among others. During this practical activity it was expected that the gene FtsZ gets successfully into the
pBKSII plasmid that would then be used to transform competent E.coli bacterial cells.
Method
Exercise 3A: Bacterial Transformation
The first approach entailed the production of competent E.Coli cells had been done. Subsequently the
process of transformation using heat-shock transformation technique was done. All the steps involving
heat–shock transformation were done under sterile environment under a Bunsen burner. First, the frozen
competent E.Coli cells got thawed in ice and then 50 μL into four microcentrifuge tubes each. The tubes
then got labeled as “Transformation A, B, C and D”. Afterwards 5 μL of litigation mixture was added into
tube marked A, then 2 μL of the purified insert added into tube B followed by the addition of 2 μL of the
purified pBSKII only to tube C. Tube D contained only 50 μL of competent cells. Subsequently, the tubes
were shaken to mix the contents and then incubated in ice for about 20 minutes. At the end of the 20
minutes, the tubes got transferred to a heating block at 42° C for strictly 90 seconds. 400 μL of Super
optimal broth plus glucose was added to the cells and mixed before being transferred into a large 15mL
sterile tube to aid in aeration. The tubes containing SOC and the cells were then incubated in the shaking
incubator at 37 degrees for duration of one hour on level 3. Afterwards 20 μL and 50 μL of the cell
suspension in Tube A containing ligation mixture and competent cells was added to pre-labeled agar
plates A1 and A2. The agar plates contained 100μg/mL ampicillin, 0.5mM IPTG and 40μg/mL X-gal.

Also, controls got prepared with 50 μL of cell suspensions from both B. C and D being transferred onto
labeled agar plates containing 100μg/mL ampicillin, 0.5mM IPTG and 40μg/mL X-gal too. A disposable
spreader was used to uniformly spread the cell suspensions onto the agar plates under the flame of Bunsen
burner then plates inverted and incubated for 18-24 hours at 370 C.
Exercise 3 B: Southern Analysis of Cloned DNA
Gel Electrophoresis
The Southern analysis began by preparation of the electrophoresis samples. A total of five tubes well
labeled got used with Tube 2-“pBKSII” containing 18 μL of MilliQ water, 4 μL of the 6x Loading dye,
and 2 μL of the sample, Tube 3 “Linear “pBKSII” contained 18 μL of MilliQ water, 4 μL of the 6x
Loading dye, and 2 μL of the sample, while Tube 4 “Insert” contained 15 μL of MilliQ water, 4 μL of the
6x Loading dye, and 5 μL of the sample, lastly, Tube 5 “Ligation” contained 10 μL of MilliQ water, 4 μL
of the 6x Loading dye, and 10 μL of the sample. The demonstrator then added 10 μL of DIG-labelled
marker on each of the gels used. The gels then got added in a sequential manner into the wells using one
lane for each sample. The sequence of sample addition was done by addition of 10 μL of pBSKSII firstly,
followed by 20 μL of linear pBKSII, then 20 μL of insert into the third available well, and lastly 20 μL of
the ligation mixture added into the fourth well. The labeled gel was then run at 160V for duration of 45
minutes to allow for complete electrophoresis to take place.
Transfer Method
The apparatus was then dismantled and the gel placed onto a plastic container and an image of the gel
taken. The DNA in the gel then got denatured by soaking it in a denaturing solution that contained 1.5M
sodium chloride and 0.5M sodium hydroxide then agitated for 30 minutes on the rocker. The gel then got
rinsed gently using water and the neutralized using the neutralization solution containing 1M Tris with a
pH of 7.4 and 1.5M sodium Chloride the agitated for about 15 minutes on a rocker. Subsequently, two
pieces of filter paper larger than the gel were laid onto an upside down container resting on a tray
containing 10X SSC (Tris-sodium citrate buffer) as demonstrated in the image below:
The gel was the placed upside down on top of the wet filter papers. Further, a piece of nylon membrane
was laid onto the gel carefully followed by two strips of filter paper soaked in 10X SSC. Subsequently, a
wad of dry paper towel was placed on top of the wet paper filters above the membrane and gel. A tile
with sufficient weight to maintain contact between all the layers was then placed on the outermost part of

the gel. The transfer was left to proceed for about one hour while consistently maintaining the level of
SSC in the bottom tray. The transfer was then dismantled and the gel labeled to indicate orientation of the
membrane relative to the original gel and the die containing DNA. The membrane then got separated
from the gel and dried between two filter papers. The membrane was then baked in an oven at 120°C for
duration of 30 minutes.
Probe labeling
During the labeling process, 3 μL of Pc-ftsZ PCR product was provided by the instructor and the added
into 13 μL of deionised water. The DNA was then denatured in a heating block at 100°C for 10 minutes
then placed on ice to cool. The 4 μL of the prime labeling mxture was added to the denatured DNA,
mixed and then placed on the centrifuged followed by one hour incubation at 37°C. The reaction was then
stopped using 2 μL of EDTA.
Hybridization Method
The hybridization step was performed by the instructor. The instructor gently transferred the membrane
already baked into the hybridization bottle. 5mL of pre-warmed DIG Easy-Hyb solution was added into
the hybridization bottle and the cap replaced tightly followed by insertion into the oven at 42°C for 30
minutes. The DIG-labelled probe was the denatured at 100°C for 5 minutes and then rapidly cooled in ice
before adding 5mL of DIG Easy-Hyb solution. The mixture was then incubated overnight at 42°C.

Results
Image 2: Analysis of transformants
Image 2 shown above, the intact plasmid and linear plasmid pBSKII did not form any bands as they
lacked the insert. On the other hand, the insert FtsZ showed a single band upon illumination indicating
efficiency of the restriction endonucleas step. Further, the ligation fragments indicated a band similar to
FtsZ indicating that the transformation process was successful.

Image 3: Colony Images
Image 3 above indicates the colony formation and characteristics from LB plates A1, A2, B,C and D after
the transformation. In plate A1, all colonies are white indicating that the E.coli cells have taken up the
insert. Plate B shows the growth of majority white colonies and a blue colony. Plate B was used as a
controls.
Discussion
The transformation of plasmid DNA into competent E.coli cells through the heat shock method is an
important molecular biology technique. The process entails combining the recombinant plasmid and the
competent bacteria. It should be noted that successful transformation of bacterial cells requires careful
handling of the cells and the heat-shock procedure. The prepared competent cells are often stored at -700C
to stop their metabolism. As such, they must be thawed to regain their metabolic activities and to take up
the plasmids (Chan et al., 2013). Care is also taken to place them on ice and prevent them from reaching
room temperature as they may lose their competency and affect the transformation process. Heat shock
process entails exposing the cells to a temperature of 420C briefly then transferred to ice. The sudden
temperature elevation leads to phase transition in the plasma membrane of the selected bacterial cells
hence affecting their selectivity integrity to facilitate uptake of the plasmids (Singh et al., 2010). The heat

shock applied induces the synthesis of DNA binding receptor proteins on the membrane of the bacterial
cells as well as translocase enzyme that imports the plasmid DNA into the cells through adhesion zones.
When the heat shock process is not done well, the transformation efficiency would be greatly affected.
During the blue-white selection process, the ampicillin-resistance and lac Z play critical roles. The plate
for selection contains both X-Gal and ampicillin antibiotic. Only bacterial cells that acquired the
recombinant plasmid would have a white colony because the restriction endonucleases cleaved the lac Z
gene sequence (Parsley et al., 2010). Lack of lac Z ensures there is no production of beta-galactosidase
hence X-gal was not cleaved to form 5-bromo-4-chloro-3-indoxyl blue product. It should be noted that
the multi-cloning site of the E.coli is located within the lac Z sequence that codes for beta-galactosidase.
Hence, the E.coli cells that successfully take up the recombinant plasmid would not be able to code for
the enzyme making the selection an easy task. The colonies that had the blue coloration contain plasmids
that did not take up the insert and hence have an intact lacZ gene. Further, it should be noted that not all
bacterial cells may contain plasmid coding for antibiotic resistance. Hence, the presence of the ampicillin
in the screening plate ensures that only those bacterial cells that have taken up the pBKS II plasmid are
able to survive and grow in the plate to form colonies (Gilliland, 2018).
Further, the cloned inserts are analyzed through gel electrophoresis together with samples from practical
one containing intact plasmid pBKS II, digested and linearized plasmid pBKS II, and the digested Pc-
FtsZ insert. The analysis done entails both gel electrophoresis as indicated before and southern blotting
technique. Southern blotting is a hybridization technique used to identify the presence of particular DNA
sequences in a mixture of similar molecules. The identification is achieved through a stepwise process
that entails gel electrophoresis, probing and finally visualization. The capillary set-up is carefully
arranged to ensure capillary actions moves water from its region of high potential to a region of low water
potential through the filter papers hence transferring DNA from the gel onto the membrane.
Subsequently, the positively charged membrane enables ionic binding of the negative DNA fragments to
it. Baking of the membranes facilitates stronger attachment of the DNA fragments to the membrane. A
probe used for the southern blotting procedure should have a complimentary sequence in the DNA sample
being probed. The practical utilized labeled DNA probes of the gene FtsZ sequence identification process.
During the preparation of the probe, it gets denatured so that the short fragments can be used effectively
for the probing. Duplex DNA cannot be used for the probing activity. On the other hand, the membrane
containing DNA fragments from the gel is hybridized by a probe to facilitate visualization of the desired
genetic sequence. The prehydization step ensures that the non-specific sites are all blocked before the
addition of the actual probe. The DIG labeled probe is denatured to ensure there are single stranded DNA
fragments that would anneal to complimentary sequences on the membrane.

PRACTICAL 4: SOUTHERN BLOT, PCR COLONY SCREEN
Introduction
In genetic manipulation processes it is always important do undertake confirmation tests due to their level
of sensitivity and high costs incurred. Similarly, for this practical session there was need to confirm the
presence of the probe and the presence of the constructs in the E.coli bacterial cells transformed through
the ligation process. During the last experiment, DNA fragments had been transferred onto a nylon
membrane. During the process, there was high probability of unspecific binding of the probes. For clarity
of the visualization process, the unspecific bound probes should be removed first. The specificity required
during the detection process relies on the level of stringency applied. Stringency in southern blotting
relates to the extent to which DNA from different sequences bind to form DNA complexes for detection.
The choice between low and high stringency depends on the matching of the targeted DNA sequences.
For instance, low stringency can be used when the DNA sequence of the probe does not match the target
sequence on the membrane. On the other hand, a high stringency requirement dictates that the sequence
of the probe should match that of the target DNA sequence on the membrane (Davis, 2012). It should be
noted that the level of stringency can be adjusted during the practical activity by adjusting the temperature
and concentrations of the detergent and salt of choice. The detection is important to determine the
efficacy and accuracy of the previous steps used to enable the transformation of E.coli cells.
Moreover, the polymerase chain reaction can be effectively bused to detect the presence of foreign
genetic materials in any organism. This is achieved through the use of primers that act as probes for the
specific desired sequences in the target genes. PCR enables amplification of small pieces of DNA
sequences that can then be visualized and hence detected. The primers used for the PCR process should
be complementary to specific target sequences in the vector selected. During this practical activity it was
expected that the southern membrane would contain the insert gene FtsZ.
Methods
Exercise 4A: Southern Blot continued
The activity began by the removal of any non-specifically bound probe onto the membrane by addition of
30mL of a stringency wash containing 2xSSC/0.1%SDS into the hybridization bottle incubated for 10
minutes in the oven at 65°C. The low stringency wash is then discarded and 30mL of high stringency
wash containing 0.5 x SSC/0.1% SDS then incubated under similar conditions as previous one for 15
minutes. The membrane was then removed from the bottle and placed into a plastic container carefully
followed by addition of 30mL wash buffer then incubated for 5 minutes at room temperature. The wash
buffer was then discarded and then the membrane placed into 20mL of antibody and incubated for 30
minutes at room temperature with slight agitation. Afterwards the antibody solution was discarded and the

membrane washed with wash buffer twice for 15 minutes each at room temperature. The wash buffer was
then discarded and the membrane equilibrated in 20mL of detection buffer for five minutes at room
temperature. The detection buffer was the removed and 30mL of color substrate solution added to the
membrane and incubated in a dark cupboard for 16 hours. The reaction was the stopped by the removal of
color substrate solution and rinsing in deionised water.
Exercise 4B: Identification of Positive Transformants
In this exercisean LB agar plate containing ampicillin, IPTG and X-gal are labeled 1, 2,3,4,5, -ve and +ve.
Subsequently, a PCR master mix was prepared as shown in the table below:
From the PCR master mix prepared above, 20 μL was transferred into seven pre-chilled 0.2mL PCR
tubes. 20 μL tip was used to pick cells from a white colony that were then transferred into respective
0.2mL PCR tubes choosing a different colony each time for all the tubes. Similarly, the tubes used to
introduce DNA into the PCR tubes was used to transfer cells into a prelabeled LB agar plate as indicated
in the image below.

A negative control was prepared by the addition of a sterile 20μL tip to the 0.2mL PCR tube. On the other
hand, positive control was prepared by touching a blue colony using the 20μL tip then dipping into a PCR
0.2mL tube as well as touching the LB agar plate with it. Subsequently, the PCR reaction was undertaken
and the LB agar plates incubated at 37°C for about 18-24 hours.
Discussion
It should be noted that DIG-labeled probes possess similar hybridization kinetics to radiolabeled probes.
Care should be taken because at times labeled probes can hybridize to sequences that show homology but
are not necessarily homologous to the probe sequence itself. However, the hybrids resulting from such
non-specific hybridization processes are not matched perfectly and are less stable. They are easily
removed by the use of washes with varying stringencies. As noted, the stringency can be varied by the use
of different salt-concentrations. The stringency can also be varied by varying the temperatures and
detergent concentration. When there is high similarity between the probe and the target gene, high
stringency must be achieved.
The identification of the bacterial cells that have acquired the plasmid DNA can also be confirmed by the
use of polymerase chain reaction. Polymerase chain reaction is a process through which genes and
fragments of DNA are amplified. The process requires the availability of a DNA template, primers, DNA
Nucleotides, Taq Polymerase and a buffer. The process normally proceeds through three key stages that
include denaturation, annealing and extension. The experiment utilized T3 and T7 primers that are
complimentary to the sequences of the plasmid vector pBKS II-PcftsZ construct. The polymerase chain
reaction detection is done concurrent to LB agar culture plates. Notably, only the white colonies are used
during the PCR and culture because they are believed to contain the pBKS II-PcftsZ construct. During the
process, both positive and negative controls are included. The negative control used is only a tip because
it contains no sample at all. On the other hand, the positive control used for this process would be cell

from the blue colony. The PCR technique is done inside a thermocyler where the temperatures can be
varied to achieve the steps of denaturation, annealing and extension (Davis, 2012).

PRACTICAL 5: SOUTHERN BLOT AND COLONY SCREEN RESULTS
Introduction
During this activity the screening process was done further. The amplified DNA molecules in the PCR
tubes are run on agarose gel through an electrophoresis to determine the formation of expected bands. The
colony PCR products are run concurrent to standards having known molecular weights to enable the
prediction of the molecular weights of the samples. Further, the screening process entails the screening of
the LB/Amp agar plates stored from the last practical sessions. Also, the results of the Southern blot
would be observed at this stage. It is the last confirmation stage to give information about the efficiency
of the molecular techniques used and accuracy, and viability of the reagents. The results can also be used
to make specified improvements for enhanced results and accuracy (Davis, 2012). It was expected that the
results of PCR and Southern Blotting would be visualized.
Method
To the prepared PCR tubes, 6x loading buffer was added to make 1x final solution concentrate. The
samples then got loaded into 1.5% agarose gel that had already been prepared and then run at 120V for 45
minutes in the electrophoresis tank. The LB agar plates also got examined recording the observations
from each of the labeled areas. The southern blot also was examined; the blot was examined for any
brown /purple bands. Further, their molecular weights got assessed using the DIG-labeled molecular
marker. At the end of the electrophoresis process, the agarose gel was observed under UV light and a
picture of the gel taken.
Results
Image 4: Confirmation of Positive cell Tranformants

According to the gel shown above, colony 4 has formed two bands while colony five has formed a single
band
Discussion
The polymerase chain reaction cannot be completed without visualization. Different technologies can be
used in the visualization process like in the case of real-time PCR where the results can be visualized
from the desktop. On the other hand, agarose gel electrophoresis can also be used in the visualization
process of the DNA bands. As such, a gel electrophoresis was set-up and the DNA samples from the PCR
screening process used as samples. The resultant bands from the gel then got visualized and a picture
taken. Similarly, the LB/ampicillin plates were viewed to examine the color and growth of the colonies to
confirm the presence of the plasmid pBKS II-PcftsZ construct. The DIG labeled molecular marker is used
in the determination of the molecular weights of the DNA fragments formed to enable their comparison
with the gene of interest Pc-FtsZ.

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