OBJECTIVE

1. To construct and analyze DNA genomic library;

2. To understand the methodology in constructing genomic DNA library and its
applications;
3. To purify, ligate and transform selected DNA fragment into selected vector; and
4. To differentiate inserted vectors from ampicilin, X-Gal and IPTG contained LB
plate.

INTRODUCTION
DNA library is a collection of cloned DNA fragments. There are two types of
DNA library which are the genomic library contains DNA fragments representing the
entire genome of an organism and the cDNA library contains only complementary DNA
molecules synthesized from mRNA molecules in a cell. The DNA library conduct in this
practical is genomic library. A genomic library is a population of host bacteria, each of
which carries a DNA molecule that was inserted into a cloning vector, such that the
collection of cloned DNA molecules represents the entire genome of the source
organism. This term also represents the collection of all of the vector molecules, each
carrying a piece of the chromosomal DNA of the organism, prior to the insertion of these
molecules into the host cells.

Partial Sau3A1 Digestion Method

Partial Sau3A1 digestion method is one of way to construct a genomic library of
overlapping fragments. Partial digestion is important to solve the problem of not
randomly distributed restriction sites. For this practical, we will use Sau3A1 as it is a
four base cutter enzyme, which will generate more fragments as it cuts frequently. It will
produce fragments of 256 bp on average. Overlapping fragments are also important in
order to connect one clone to another in the library. Partial digestion helps to produce
more and better cut of DNA fragments either in variation or number. (Dale &Schantz,
2002)
Phenol-Chloroform Extraction
Phenol-Chloroform extraction is a method used to purify RNA or DNA. It can be
done either by using only phenol or phenol/ chloroform. Phenol consists of a solution
that is actually about 72% phenol, 28% water. Since phenol is a weak acid, the
solutions that we use have been equilibrated with buffer to bring the pH to a particular
target – either acidic for RNA purification or slightly alkaline for DNA purification.
Chloroform is significantly denser than water as the presence of it in the organic phrase
increases the overall density of the organic phrase, thus, helping to prevent phrase
inversion. Furthermore, the chloroform help reduce the interphase. Interphase is the
fuzzy border between the two phrases. Interphase may have partially denatured
proteins, DNA (depends on pH), and/or partially denatured DNA binding protein which
still clinging to DNA. Therefore, interphase may influenced the result as it may cause
the presence of the residues. Chloroform cannot be used alone because chloroform is
not a good organic solvent when works with the environment with proteins as in our
practical. Thus, chloroform should be used after the phenol had been added. It function
to further denatures proteins or DNA (depends on pH) and also removed the phenol.

Ethanol precipitation of DNA

Ethanol precipitation is a commonly used technique for concentrating and de-
salting nucleic acid (DNA or RNA) preparations in aqueous solution. The basic
procedure is that salt and ethanol are added to the aqueous solution, which forces the
nucleic acid to precipitate out of solution. The precipitated nucleic acid can then be
separated from the rest of the solution by centrifugation. The role of the salt in the
protocol is to neutralize the charges on the sugar phosphate backbone making the
molecule far less hydrophilic, and therefore much less soluble in [Link] is much
less polar than water. If enough ethanol is added electrical attraction between
phosphate groups and any positive ions present in solution becomes strong enough to
form stable ionic bonds and precipitate DNA. Resuspend DNA fragment in TE buffer,
the purpose of TE buffer is to protect DNA or RNA from degradation.
Ligation of digested genomic fragments into pUC 18 cloning vector.
In this practical, the DNA fragment digested by Sau 3A1 will then insert into Bam
H1-digested plasmid vector (pUC18). The ligation between vector and DNA fragment
will use T4 DNA Ligase. Although different restriction enzymes are used to cut the DNA
fragment and vector, both of them produce sticky ends that compatible to each other.
The recognition site for Sau 3A1 is /GATC and for BamHI is G/GATCC. A vector is a
DNA molecule used as a vehicle to transfer foreign genetic material into another cell.
pUC18 is a plasmid cloning vector commonly used with E. coli. pUC18 is the
improvement of pBR322 which the construction is simplified by the use of polylinkers or
multiple cloning site (MCSs) (Vieira & Messing 1982, 1987, Yanisch-Perron et al, 1985).
The MCS is inserted into the lacZ’ gene but it does not interrupt the gene function. MCS
extends the range of enzyme that can be used to generate restriction fragment for
cloning, thus increasing the number of potential cloning strategies. pUC18 also has beta
lactamase (bla) an ampicilline resistance which acts as selective marker, origin of
replication (ori) and lacI encode for repressor of lac promoter. The vector length is2686
bp and is isolated from E. coli strain DH5α by standard procedures.

Ligation is a process of creating a phosphodiester bond between the 3' hydroxyl

of one nucleotide and the 5' phosphate of another T4 DNA ligase is an enzyme that is
encoded by the bacteriophage known as T4. T4 DNA ligase is mostly used in the joining
of DNA molecules with compatible cohesive termini or blunt-ended, double-stranded
DNA to one another, or to synthetic linkers. The unique T4 DNA Ligase buffer optimizes
ligation which can be performed in 5 minutes. T4 DNA Ligase also requires ATP as
cofactor. Thus, it is important to use phosphorilated vector in order to provide ATP to
the enzyme (Primrose & Twyman, 2009).

Bacterial transformation
Transformation is the process by which the genetic material carried by an
individual cell is altered by the incorporation of foreign DNA such as plasmid vector into
its genome” ([Link], “Definition of Genetic transformation”). Transformation
in bacterial cells occurs when the cell incorporates naked DNA into its genetic material.
If the foreign DNA has an origin of replication recognized by the host cell DNA
polymerases, the bacteria will replicate the foreign DNA along with their own DNA.
When transformation is coupled with antibiotic selection techniques, bacteria can be
induced to uptake certain DNA molecules, and those bacteria can be selected for that
incorporation. Bacteria which are able to uptake DNA are called "competent". [Link]
does not exhibit natural competence. Thus, the competence in [Link] is induced by
washing them with ice-cold calcium chloride. The bacterium used in this practical is
[Link] which grow in human intestine. Because they grow in humans, they will grow best
at human body temperature (37°C), thus the culture need to be incubated in 37°C.
Lysogeny broth (LB), a nutritionally rich medium, is primarily used for the growth
of [Link] continues to be one of the most common media used for maintaining and
cultivating recombinant strains of Escherichia coli. Ampicillin is an antibiotic. One of the
genes in the plasmid codes for the ampicillin resistance protein, and thus will allow
bacteria with the plasmid DNA to grow in the presence of ampicillin. X-gal is an organic
compound consisting of galactose linked to a substituted indole. X-gal used to indicate
whether a cell expresses the β-galactosidase enzyme, which is encoded by the lacZ
gene, in a technique called blue/white screening. IPTG induces synthesis of ß-
galactosidase, an enzyme that promotes lactose utilization and it is used to induce Lac
Z gene expression.
The mixture is first cooled in ice in order to reduce the movement of the
phospholipids in the cell wall, allowing for the plasmid to pass through more easily. The
heat shock, in a sense, catalyzes the transfer of the plasmid into the bacterial cell by
both expanding the pores of the cell and providing the plasmid with the energy to
penetrate the cell. X-Gal was used to modify the galactose sugar that is metabolized by
β-galactoside to form an insoluble product. IPTG induces synthesis of ß-galactosidase,
an enzyme that promotes lactose utilization. It is used to induce Lac Z gene expression
in cloning practicals (Primrose & Twyman, 2009).

MATERIALS
A) Partial Sau 3A1 Digestion Method
a. 200 µL of genomic DNA
b. 120 µL of 10x reaction buffer
 c. 880 µL of sterile ddH2O
B) Phenol-Chloroform Extraction
a. Phenol
b. Chloroform
C) Ethanol Precipitation of DNA
a. 3M NaAce
b. Ice cold 100% EtOH
c. 500 µL of ice cold 75% EtOH
d. 20 µL TE Buffer (pH 8.0)
D) Ligation of digested genomic fragments into pUC 18 cloning vector
a. 5 µL of DNA
b. 5 µL phosphorilated pUC 18 vector
c. 5 µL 2x T4 ligation buffer
d. 1 µL T4 DNA ligase
e. 4 µL 5x DNA dilution buffer
E) Bacterial transformation
a. 70 µL-100 µL of ice cold fresh competent E. Coli
b. 1.0 ml of L-Broth
c. LB plates containing 50 µL/mL of Ampicilin, X-Gal, IPTG

APPARATUS
Sterile tubes, vortex machine, pipette, water bath, 1% agarose gel electrophoresis,
Microtube, 10p pipette, and incubator, Micro centrifuge and adaptors, Micro pipette and
tips, parafilm, ice/ice water bath, shaker at 37 oC.

PROCEDURES

For this practical, there are 5 stages of procedures that must been carried out
which are the partial Sau3AI digestion method, phenol-chloroform extraction, ethanol
precipitation of DNA, ligation of digested genomic fragments in pUC 18 cloning vector
and bacterial transformation. Under certain circumstances, the second and third
procedure; the phenol-chloroform extraction and ethanol precipitation are not been
carried out. However, both of the procedures are important in constructing the genomic
library.

The first procedure is the partial Sau3A1 digestion method. 120 µg of Genomic
DNA has been partially is digested with Sau3A1 as followed:

Genomic DNA = 200 µL

10x reaction buffer = 120 µL
ddH2O ( Sterile ) = 880 µL
Total overall = 1200 µL

Then, the reaction is mixtured by using a vortex. After that, the reaction is alquoted into
5 sterile tube. 10 units of Sau 3 A1 enzymes then are added into tube no.1, mix the tube
by vortex and 200 µL of reaction is transfered into tube no.2 as followed by soft
vortexing. Then, 200 µL of reaction is removed and transfered into tube no.3 as followed
by soft vortexing. From tube no.3, 200µL of the reaction then is removed again and
transfered into tube no. 4 as followed by vortexing. 200 µL of the reaction then is
transfered from tube no.4 into tube no.5. After that, all the tubes are incubated at 37 0C
of waterbath for 45 minutes. The digestions are checked by using 1% of agarose gel
electrophoresis. The fraction is selected that give to us 1-5 kb fragments of DNA. At the
last, the step is proceeded with Phenol-Choloroform extraction (to deactivate the
enzyme) and Ethanol precipitation.

Next is the Phenol-Chloroform extraction, 1 volume of phenol is added into the

DNA suspension followed by vortexing for 2 minutes. The DNA suspension is spun at
5000 rpm for 5 minutes. The upper phase is removed and placed into a new sterile
tube. The same volume of chloroform is added into the DNA suspension. The
suspension is vortexed again for 2 minutes followed by spinning at 5000 rpm for 5
minutes. The upper phase is removed into a new tube. The chloroform extraction step is
repeated and the upper phase is removed into a new tube.

After that is the ethanol precipitation of DNA. 0.1 voume of 3M NaAce and 2
volume ice cold 100% EtOH are added into the DNA suspension. The EtOH~DNA
suspension is mixed slowly by swirling the tube 4-5 times. The suspension is incubated
at -70oC for 10 minutes. The tube is spun at 13000 rpm for 20 minutes. The supernatant
is removed. 500 µL of ice cold 75% EtOH is added for washing process. The
suspension is spun again at 13000 rpm for 20 minutes. The supernatant is removed and
the pellet is dried at room temperature for 15 minutes. The DNA fragments are
resuspended in 20 µL TE buffer pH 8.0

The next step is the ligation of digested genomic fragment into pUC 18 vector. 5
µL of DNA, 5 µL Phophorilated Puc 18 vector, 5 µL 2x T4 ligation buffer, 1 µL T4 DNA
Ligase and 5x DNA dilution buffer are added together. Then, the ligation mixture is
incubated at room temperature for 15 minutes. After that, the ligation mixture is
proceeded with bacterial transformation at the next day.

Finally is the bacterial transformation. Plasmid DNA or ligation mixture is added

into 70 µL-100µL of ice cold fresh competent [Link]. Then, the cells are incubated on
ice for 30 minutes. After that, the cells are heat-shocked for 2 minutes at 42 0C and are
then put on ice again for 2 minutes. 1.0 mL of the L-Broth is added and the culture is
incubated at 37oC for 1 hour. 100.0 µL of the culture is then spread onto LB plates
containing 50 µg/mL of Ampicilin, X-Gal, IPTG and incubated at 37 0C overnight.

RESULT

The actual result with the presence blue and white spots.
This is the result of our group. There are no blue or white spots on the bacterial colony.
It shows that our construction of genomic library is a failure.

DISCUSSION

Genomic library involve gene cloning strategies. There are several steps in
producing a genomic library. For this practical, the first step is to partial separation by
restriction enzyme Sau3AI, followed by phenol-chloroform purification, and ethanol
precipitation. These three steps are important to provide the fragments of genomic DNA
which have been cut by Sau3AI. The genomic library production is continued by ligating
the DNA fragments with the vector, phosphorilated pUC18. The procedure is followed
by the process of transformation and screening in order to construct and evaluate the
genomic library. Thus, it is important to follow the precautions of each step in order to
get successful genomic library. The successful DNA recombinant can be detected by
blue/white screening on the growth medium containing Xgal and Ampicillin. Based on
our result, it is a failure as there are no blue and white spots, which indicate the failure
to produce DNA recombinant. Each step is important. A slight mistake when performing
the practical will give result to the whole. In this discussion, we will highlight three main
procedures which are the partial digestion, ligation and bacterial transformation.
The first important step is the partial digestion by enzyme. This step is vital as
this is the step where the overlapping fragments are to be produced. The overlapping
fragments are used to connect the information in the library and to ensure that the DNA
fragments have uniform base pairs. However, there are some problems that might be
arising which can cause failure to our result. In partial digestion, some sites may be cut
more efficiently and rapidly than others. Besides that, the regions of the genome might
not equally represent in the library. There is also some possibility that some regions of
the genomes that over represented and other regions will occur less frequent in the
library. Hence, care must be considered and suitable restriction enzyme must be
chosen to produce better yields of DNA fragments (Dale &Schantz, 2002).

The next important step is ligation. Failure to get the result can also be related to
failure to ligate the DNA fragments and the vector. A number of ligation reaction
parameters are important, including DNA concentration, the molar ratio of insert to
vector, temperature, buffer composition, and enzyme concentration. Because ligating
blunt ends is less efficient, the process requires higher concentrations of enzyme and
DNA, a longer incubation time, and a lower reaction temperature than are used for
cohesive ends. There are errors while doing this step. The error detected is using of
excess DNA ligase during ligation. This step will affect the process of ligation. The
exceptionally high cyclization efficiency reported is correlated with the higher than
normal T4 ligase concentration used in the measurements. Besides that, the DNA ligase
will act more efficiently to smaller fragments. Failure to get proper length of DNA
fragments during partial digestion also can lead to the failure of this process (Dale
&Schantz, 2002).
In this practical, we use competent [Link] to increase the efficiency of
transformation. However, a study done in E. coli found that transformation efficiency
declines linearly with increasing plasmid size. Thus, only small levels of DNA can be
taken up by [Link]. Thus, we may assume that, if there is failure during partial digestion,
in which the DNA fragments produced are not in uniform size, there will be DNA
fragments that too big (contain too many base pairs) and when the fragment
successfully ligate to the vector, it will cause the DNA plasmid to increase in size. The
failure of transformation also might caused by the failure in DNA ligation which depend
on the ligase activity (Fermentas Life Science). The level of ampicillin resistance must
also need to be taken into consideration. High level of ampicillin resistance may affect
the transformation as ampicillin exerts its effect on cell-wall biosynthesis only in cells
which have progressed into active growth. Other possible source of error in our practical
includes insufficiently mixing the solutions of DNA after adding restriction enzymes or
not returning the mixtures to ice quickly enough after during the heat shock.

CONCLUSION
From this practical, we are able to construct and analyze the DNA genomic library by
understanding the methodology in genomic DNA library construction. In order to get
successful DNA genomic library, we should know the appropriate restriction enzyme
and vector used. The proper way and efficiency in ligation, bacterial transformation and
screening ensure the successful rate of genomic library construction.

REFERENCES

1. 1. Dale, J.W., Schantz, M.V. (2002). From Genes to Genomes,England: John

Wiley and Sons.
2. Sambrook, J. and Russel. D. W. (2001). Molecular Cloning: A laboratory Man,
New York Press, Cold Spring Harbour (3rd ed), Cold Spring Harbour
3. Primrose S.B. and Twyman R.M. (2009). Principles of Gene Manipulation and
Genomics (7th ed), Blackwell Publishing
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