This document summarizes key aspects of cloning vectors and techniques. It discusses multiple cloning sites that contain unique restriction enzyme sites used to insert DNA fragments. Both plasmid vectors and bacteriophage lambda can be used for cloning, with lambda able to accommodate larger inserts up to 25kb. The document compares features of plasmid and lambda cloning, such as circular vs. linear DNA, transformation vs. transfection, and plating methods. It also explains the transformation process, including factors that influence efficiency like calcium chloride treatment and host bacterial strain.

1. 1
Cloning
MULTIPLE CLONING SITE
 Many cloning vectors contain a multiple cloning site or polylinker:
- a DNA segment with several unique sites for restriction endo-
nucleases located next to each other
 Restriction sites of the polylinker are not present anywhere else in the
plasmid.
 Cutting plasmids with one of the restriction enzymes that recognize a
site in the polylinker does not disrupt any of the essential features of
the vector .
 MCSs let a molecular biologist insert a piece of DNA or several
pieces of DNA into the region of the MCS.
 This can be used to create transgenic organisms, also known
as genetically modified organisms (GMOs).

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 Gene to be cloned can be introduced into the cloning vector at one
of the restriction sites present in the polylinker

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◘ LacIQ
: is a repressor protein to Lac Z gene , lac repressor binding to
the operator sequence causing inactivation to lac Z gene .
- In platting system I cannot detect recombination .
◘ isopropylthiogalactoside (IPTG) : Inducer protein to Lac Z
gene , which binds to the lac repressor (LacIQ)
protein and
inactivate repressor protein , causing induction to Lac Z gene .

4. 4
BACTERIOPHAGE LAMBDA (λ - phage )
• is a bacterial virus, or bacteriophage, that infects the
bacterial species Escherichia coli (E. coli).
• The phage particle consists of a head (also known as
a capsid)(protective coat ), a tail, and tail fibers.
• The head contains the phage's double-strand
linear DNA genome.
• cloning limit : accommodate to 25 kbp of Insert .

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◘ Steps of Cloning Using λ phage :
1- Preparation :
• Lambda viral genome : linear DNA with ssDNA "sticky end" at both
ends; these ends are complementary in sequence and can hybridize to
each other (this is the cos site: cohesive ends).
• preparation The Host (E. coli)
2-Insertion :
• coupling happen in the center portion .
• Viral linear DNA have 2 arms .

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3- Packaging :
• using Capsid protein and processing Enzymes and Recombined Phage
DNA in Test Tube .
4- Transfection = Infection :-

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1- The Virus attached to The Host cell ( Attachment to the surface )
2- Inject the Viral genome ( Genatic material ) to the Host cell .
3- Replication of the viral genome ( recombinant Phage DNA ) ,
forming a new capsid and new viral genome and synthesis of new
phages ( more copies ) causing lysis of The Host cell ( E.coli ) .
5- Growing or Plating :-

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• Bacterial lawns can be produced manually by evenly spreading a
high amount of bacteria onto an agar plate .
- Bacterial lawn is a term used to describe the appearance
of bacterial colonies when all the individual colonies on a petri-
dish agar plate merge to form a field or of bacteria.
- Bacterial lawn : is a continuous Sheet of bacterial growth and a
clear area.
- clear area : clear spots in lawn called plaques .
• A clear plaque will be formed if the host is completely susceptible
to the phage ( lysis by the phage )
• After formation of the plaques , we isolate and screening the
plaques in a new agar plate .

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Comparison Between Plasmid & λ- Phage
Plasmid λ- Phage
1- Genetic
material
It is a circular double stranded
DNA
double-strand
linear DNA genome
2- Bacteria
penetration
Transformation Transfection
3- Types PBR322 , PUC 18 , PUC 19 λ- Phage
4- Protective coat Without Protective coat With Protective coat
( Capsid )
5- Size of Insert 10000 bp or 10 kb 25000 bp or 25 Kbp
6- Penetration
Steps
1- preparation
2- Insertion
3- Transformation
4- Screening
5- growing & Isolation
1- preparation
2- Insertion
3- Packaging
4- Transfection =
Infection
5- growing or Plating
7- Position Of
Insert in Vector
• insert a piece of DNA or
several pieces of DNA into
the region of the MCS.
• The Insert compliment with
the Vector at MCS .
• coupling between
insert and vector
happen in the center
portion of the vector
genome .
8- plating system • Ampicillin Agar plate
• Tetracycline Agar plate
•( X-gal ) Lac Z Agar plate
• Lawn Plate .
9- Isolation &
Growing
( Multiplication )
• The desired recombinant
colonies can be picked and
cultured , then Isolate the
Desired genome and the
vector after multiplication
• After formation of
the plaques (
multiplication ), we
isolate it and screening
the plaques in a new
agar plate , then
isolate the desired
gene from the vector .

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Transformation
◘ Definition :
• Transformation :- Insertion the Vector as recombinant DNA or Non-
recombinant DNA into a Host cell ( E.coli ) .
• Transformation :- when microorganism are able to take up and
replicate DNA from their local environment .
◘ The condition of insert the vector into the Host cell :-
1- Change the permeability of the Host cell wall by using ions like
calcium – chloride that change the structure of the cell wall and increase
permeability of the host so as to let the vector enter the cell wall of the
host.
• Or by using Electroporation :- use high electrical voltage pulses to
translocate DNA across the cell membrane .
2- Temperature of Host cell must be very low , if the temperature
increase the transformation process cannot be done .
3- Increase the number of host cell ( ex : Bacteria )
4- The Size of the vector ( plasmid ) the more smaller the easy to
enter the host cell .
5- The type of ( E. coli ) ( The Host ) :
- RecBc gene : make expression for an enzyme called exonuclease
that used to cut any linear foreign DNA .
cut only linear if the DNA is circular there is no effect on it .

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1- RecBc
+
gene : Recombinant Bacterial cell gene ( the gene is
present )
- If the bacteria have RecBC+ gene it will transform the circular
DNA 100 % but , if the DNA is Linear it will transform it only 10
% .
2- RecBc
-
gene : Recombinant Bacteria cell
- if the gene is absent , transformation of the linear and circular
DNA 100 % ( completely transformed ) .
◘ Cos – Sequence : (cohesive ends )
5՝ cos sequence Central portion 3՝ cos sequence
Order the recombinant genome to ( packaging ) and formation the capsid
around it before transformation .
The lecture is Done ♥ ☺ Q.A