Cloning pasting into

vectors and
transformation

Cloning

Vector
Fragment of DNA
Recombina
nt vector

Clone(s)
Host

Vectors
Carriers of DNA transfer and replicate
inserted DNA
Plasmid circular dsDNA
In nature: contain non-essential genes
In the lab: highly modified and engineered to be easier to
manipulate and contain useful components

Plasmid
vectors
Circular

Plasmids can be used

as vectors

Useful restriction sites for cloning into vectors are

Unique only one recognition sequence in the whole
plasmid
Clustered many different recognition sequences close
together in a multiple cloning site (polylinker)
EcoRI BamH HindIII KpnI
I

EcoRI

Plasmid
vectors
Circular
Multiple
cloning site

BamH
HindIII
KpnI
I
5 ACGGAATTCAAAGGATCCTTTAAGCTTAAAGGTACCACG
3
3 TGCCTTAAGTTTCCTAGGAAATTCGAATTTCCATGGTGC 5

Ligating DNA into a

plasmid

The DNA ends must be compatible

EcoRI

BamH
HindIII
KpnI
I
5 ACGGAATTCAAAGGATCCTTTAAGCTTAAAGGTACCACG
3
3 TGCCTTAAGTTTCCTAGGAAATTCGAATTTCCATGGTGC 5
EcoRI
5 ACGG
AATTCAAAGGATCCTTTAAGCTTAAAGGTACCACG 3
3 TGCCTTAA
GTTTCCTAGGAAATTCGAATTTCCATGGTGC 5

Ligase
5 ACGGAATTC
3 TGCCTTAAG

5 AATTC
3
G

G
3
CTTAA 5

GAATTCAAAGGATCCTTTAAGCTTAAAGGTACCACG 3
CTTAAGTTTCCTAGGAAATTCGAATTTCCATGGTGC 5

Ligating a PCR
product with a
plasmid

Because PCR primers are incorporated into

the product, you can use primers to add
sequences to products

5 TGGGCGCCCCACCACGCCTCATGTGTGACGCCTGCAGGACAGGGGACAGATGACCA 3
CGTCCTGTGGGGTGTCTCTT
5G
AAG
AAT
5
TC
GCCCCACCACGCCTCATGTG
3 ACCCGCGGGGTGGTGCGGAGTACACACTGCGGACGTCCTGTCCCCTGTCTACTGGT 5
PCR
GAATTC
CTTAAG

GAATTC
CTTAAG

EcoRI
AATTC
G

G
CTTAA

Ligase

DNA ligase
Compatible ends come together, sealed by
DNA ligase
In nature: joins Okazaki fragments

DNA ligase forms covalent bond to seal

backbone

Ligase only joins

compatible ends

Sticky ends are compatible if the overhangs

are
fully complementaryLigase

GATCCGT3
5AAAG
GCA5
3TTTCCTAG

5TCAG
3AGTCCTAG

5AAAGGATCCGT3
3TTTCCTAGGCA5

A T C C G T 3 Ligase
GCA5

5TCAG ATCCGT3
3AGTCCTAGGCA5

Blunt ends are always compatible (but not

very sticky)

5GTAG
3CATC

CGT3
GCA5

Ligase

5GTAGCGT3
3CATCGCA5

Transformation
Plasmids are transformed into competent
bacteria (able to take up DNA from the
environment)
Heat or
electricit
y

Competent
cells

Plasmid
vectors
Circular
Multiple
cloning site
Small

Recovery &
growth

Each colony arises from a single bacterial

cell therefore all cells in a colony are
genetically identical these cells in each
colony are a clone

Plasmid replication in
the host

If there is just one plasmid in a cell how

does it share?
Therefore plasmid needs an origin of
replication (ori)
MCS
V for
vegetative
allows many
copies

oriV

Plasmid
vectors
Circular
Multiple
cloning site
Small
ORI

Selecting for
transformants

Not all bacteria will be transformed

Non-transformant

Competent
cells

How to tell the

difference
between
transformants
and nontransformants?

Transformant

Selecting for
transformants

Ampicillin
(an antibiotic) kills bacteria
-lactamase
breaks down ampicillin
t:
an
- orm
on sf e
N an itiv
tr ns
se

an
m
r
o
sf
Tran istant
res

t:

Media
containing

Selecting for
transformants

Selectable markers genes that can produce

easily observed characteristics

ampR

Plasmid
vectors
Circular
Multiple
cloning site
Small
ORI
Selectable
marker(s)

E.g. the ampR gene can be transcribed and

translated by the host to produce lactamase

Selecting for plasmids

with inserts
Competent
cells

How do we ensure the plasmid contains an

inserted piece of DNA (recombinant)?
MCS (within lacZ
gene)
lacZ
ampR

oriV

Plasmid vectors
Circular
Multiple cloning
site
Small
ORI
Selectable
marker(s)

Selecting for plasmids

with inserts
Non-recombinant

lacZ gene (screenable marker) encodes galactosidase

Xgal

Recombinant

Blue compound

Insertional
inactivation
Beta-galactosidase crystal image from Wikimedia

Media
containing

Checking for inserts in

Colony PCR plasmids
Pick colony

Replica plate

Add to PCR tube

and amplify

Checking for inserts in

plasmidsGrow small-scale Purify
Restriction digestion
Pick colony

Replica plate

culture

plasmid
DNA

Checking for inserts in

Sequencing plasmidsGrow small-scale Purify
Pick colony

Sequence
DNA

Replica plate

culture

plasmid
DNA

Two real-life vectors

Plasmid vectors
Circular
Multiple cloning
site
Small
ORI
Selectable/screena
ble marker(s)
http://www.fermentas.com/templates/files/tiny_mce/family_images/sd004_fam.gif and

Cloning

Selected fragment
of DNA
+ vector
= recombinant
vectors
+ host

=
clone(s)

Learning objectives
Determine and manipulate sequences in relation
to molecular operations on DNA (e.g. digestion
and ligation)
Identify and explain the components of cloning
vectors and extrapolate the function(s) and
action(s) of these components