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Objectives
• describe the steps in gene cloning by using
plasmid as the vector
1
The steps for cloning:
1) Isolation
2) Splicing
3) Insertion
4) Transformation
5) Screening
Steps in Gene Cloning
1. Isolation of DNA (gene)
2. Slicing with restriction enzymes
3. Insertion
4. Transformation & amplification (multiplication
@ cloning)
5. Screening ; to identify bacterial cell containing
recombinant plasmid
Probing ; to identify bacterial cell containing
recombinant plasmid with target gene
3
Isolation
 isolation of plasmid DNA (from E. coli) and
DNA containing gene of interest
- plasmid DNA as cloning vector
♫ plasmid DNA carries ampR gene and lacZ gene
- ampR gene: antibiotic resistance gene
- lacZ gene : encode for β-galactosidase 
4
Isolation
5
Slicing (cutting / cleave)
 cut open the plasmid DNA at restriction site
which lies within lacZ gene
 cut out the DNA into many
DNA fragments; some of these fragments carry
the gene of interest
- by using same restriction enzyme which cut
at restriction sites containing same palindromic
sequence to produce sticky ends
6
Insertion
 after mixing, the DNA fragments and the
cut plasmids form the complementary
pairs
 they are then
joined by DNA ligase

creating a mixture
of rDNA molecules
♦
note that the lacZ has become
nonfunctional
♦
cannot code
for β-galactosidase
Transformation
 bacteria containing recombinant plasmid CAN’T
produce β-galactosidase

the rDNA are reintroduced into the bacteria

bacterial cells are mixed with rDNA
in the presence of cold calcium chloride

followed by heating; making the bacterial
cell wall permeable to plasmids
Transformation
Transformation
 produce diverse of bacteria :
• bacteria with recombinant plasmid (containing
gene of interest) e.g. gene encode for insulin
• bacteria with recombinant plasmid (containing
gene which encode for other protein)
e.g. gene encode for human growth hormone
• bacteria with NON-recombinant plasmid
13
Screening
Procedure 1 Blue-white screening
i.e. detecting the bacteria containing rDNA
Blue-white screening
- to identify bacteria containing recombinant plasmid
 bacteria is cultured on medium containing
antibiotic (ampicillin) and sugar (X-gal)
Observation
ONLY bacteria with plasmid grow ; has ampicillin
resistance (ampR) gene
15
X-gal is used to identify colonies bacteria with
recombinant plasmids

bacteria colonies WITHOUT recombinant
plasmid will stain blue ;
β-galactosidase is produced by functional
lacZ gene (hydrolyze X-gal to yield blue product)

bacteria colonies WITH recombinant plasmid
will stain white ;
β-galactosidase is NOT produced because
lacZ gene is NON-functional; X-gal is NOT
hydrolyzed
16
Probing (Nucleic acid hybridization)
Probing (Nucleic acid hybridization)
- to identify bacteria with recombinant plasmid
containing gene of interest (target gene)
 based on base pairing between gene of
interest (e.g. gene encode for insulin)
and other DNA molecule known as DNA probe
(short & single-stranded; labeled with radioactive
isotope or fluorescent tag)
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Probing (Nucleic acid hybridization)
STEP 1
 a master plate is prepared

- contain colonies of bacteria with recombinant
plasmid
in the mean time, DNA probe is prepared
STEP 2
 nitrocellulose filter paper is placed onto the

master plate
the filter paper is pressed against the bacterial
colonies on the master plate ; bacterial colonies
is transferred onto the filter paper
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Probing (Nucleic acid hybridization)
STEP 3
 the filter paper is treated with NaOH or heat
- to denature (separate) the DNA;
double helix DNA  single stranded DNA
STEP 4
 DNA probe solution is added to the filter paper
- DNA probe will hybridize (base-pair with
any complementary bases of single
stranded DNA)
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Probing (Nucleic acid hybridization)
STEP 5
 the filter paper is washed to remove the excess,
unbound DNA probe
 then, the filter paper is laid on X-ray film
– autoradiography technique
 the film (autoradiograph) is developed
STEP 6
 the autoradiograph is compared with the

master plate
the colonies which contain gene of interest
is identified
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21
REMIND AGAIN
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Isolation
Slicing
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Insertion
24
Transformation
Screening
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Probing
STEP 1
Master plate
DNA probe
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Probing
STEP 2
Contain NAOH or Heat
To denature DNA ,double
to single
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Probing
STEP 4
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Probing
STEP 5
STEP 6
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The steps for cloning:
1) Isolation
2) Splicing
3) Insertion
4) Transformation
5) Screening
Screening
Blue-white screening
i.e. detecting the bacteria containing rDNA
Probing (Nucleic acid hybridization)
- to identify bacteria with recombinant plasmid
containing gene of interest (target gene)
40