Postpartum Complications Ahmed Ali MS, PhD Dept. Theriogenology, Assiut Univ., Assiut, Egypt.
Detection of your cloning and - Assiut University from gene to...Phenotypic screening is used when...
Transcript of Detection of your cloning and - Assiut University from gene to...Phenotypic screening is used when...
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Detection of your cloning and
pick up your right clone
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Introduction Once recombinant plasmid is constructed, it is introduced
into recipient cells.
Introduction of recombinant DNA into recipient cells is
called transformation: introduction of foreign DNA
changes (transforms) properties of the organism.
Special treatment makes cells competent - capable of
accepting foreign DNA.
Usually, these treatments make cell membrane more
permeable for a DNA molecule.
When competent cells are mixed with DNA some cells
(actually, very few) become transformed.
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Competent cells transformation
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Competent cells culture:
After transformation, cells
are plated onto agar
medium that contains
selective antibiotic: only
transformed cells, will
survive and form colonies.
All the untransformed
cells will die.
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In each colony formed on the agar plate, all cells are
descendants of one transformed cell.
All cells in the clone are genetically identical and contain
the same recombinant vector
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How to Find and Pick Up the Right
Clone?
The most common methods include:
1. Phenotypic screening.
2. Screening with antibodies.
3. DNA hybridization.
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Phenotypic screening
Phenotypic screening is used when cloned gene is
expressed and changes properties of the cell in an
"obvious way". E.g. Blue-White Screening
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Blue-White Screening pDrive expresses LacZ α-peptide Provide β-galactosidase activity. X-gal is a
colourless analog of lactose cleaved by β-galactosidase to form a bright blue
insoluble pigment.
No insert intact lacZ Blue
Insert disrupted lacZ White
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BLUE – WHITE SCEENING
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BLUE – WHITE SCEENING
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Screening with antibodies
Screening with antibodies is used when cloned gene is
expressed and antibodies recognizing the encoded
protein are available
(Antibiotic Resistance).
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Confirmation
By using PCR:
The same primer
to detect the
insert
Specific primer
for vector
detection
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PCR for detection of the vector which
carry the insert:
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DNA hybridization
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DNA hybridization
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DNA hybridization
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DNA hybridization
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DNA hybridization
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Thanks a lot with my Best Regards and My Best wishes
Amira A. AL-Hosary
E-mail: Amiraelhosary @yahoo.com
Mob. (002) 01004477501