Molecular Cloning
Biology 20L
Spring 2003
Overview of Molecular Cloning
• Restriction digest of plasmid pUC19 and phage 
– GOAL: Linear pUC19 DNA and several fragments of
phage  DNA
• Ligation reaction
– GOAL: pUC19 recombined with one or more 
fragments
• Transformation reaction
– GOAL: Use a bacterial host create multiple copies of
our new DNA construct (cloning)
Overview of Molecular Cloning
• Identification of candidates
– GOAL: Determine which bacteria have the desired
product (plasmid pUC19 with  fragment insert)
• Re-isolation of plasmid DNA
– GOAL: Obtain multiple copies of the desired DNA
construct
• Restriction map of plasmid DNA
– GOAL: Determine which  fragment was inserted in
pUC19
Restriction Digest of plasmid pUC19 and phage 
• Use the restriction enzyme Hind III to cut both plasmid pUC19 and
phage .
• The recognition sequence for Hind III is: A•AGCTT. This specific
sequence occurs once in pUC19, and occurs seven times in phage .
• One site in pUC19 creates one linear piece of DNA when cut.
• Seven sites in  create 8 fragments when cut.
• The DNA fragments will be separated and analyzed with gel
electrophoresis.
•BstEII pUC19 pUC19 •HindIII  •HindIII •BstEII
pUC19 a genetically engineered plasmid
• 2.7 Kb (small size allows lots of room for inserting DNA)
• Circular non-genomic DNA.
– Phage  DNA is linear and much larger (48.5 Kb).
• Has an origin of replication, and a high copy number. (200+/cell)
• Ampicillin resistance gene
– Codes for an enzyme that binds and degrades ampicillin.
• Lac Z gene (part of the Lac operon)
– Codes for ß-galactosidase
– An enzyme that breaks down lactose into glucose and galactose.
– Polylinker cloning site within LacZ
• Contains recognition sequences for several restriction enzymes.
• A disruption at this site prevents the production of ß-galactosidase.
http://www.fermentas.com/techinfo/NucleicAcids/mappuc1819.htm
T4 Phage
T4 phage infecting a bacterial cell
Ligation Reaction
• Hind III breaks covalent bonds at the recognition sequence.
– A•AGCTT
– T TCGA•A
A
TTCGA
AGCTT
A
• The complimentary or “sticky” ends can readily form H -bonds.
• During the ligation reaction, the linearized plasmid and  fragments
are combined.
– Most of these DNA fragments have “sticky” ends. However, because  is
originally linear, the fragments cut from each end do not have overhangs
complementary to the HindIII cuts.
• When combined, the pieces form H-bonds in various configurations.
• The enzyme DNA ligase is used to form new covalent bonds.
– ATP drives this reaction.
Some possible ligation reaction products:
Recombinant
No insert
Fragments
No ligation
Transformation
• Using bacterial cells to amplify the DNA of interest.
• Competent cells are able to take up foreign DNA and acquire genetic
information.
• Ordinary Escherichia coli cells can be made competent through a
treatment with Ca2+ .
– Competent cells have very fragile cell walls, and must be handled gently.
• During the transformation reaction:
– Competent cells are combined with the ligation products.
– Incubated on ice (DNA sticks to the outer cell walls.)
– Heat Shocked (Membranes become more porous and allow DNA to enter.)
• Not all the competent cells will take up DNA. We will determine the
frequency.
– Incubated in LB broth at 37ºC for ≈ 45 min.
• Long enough to allow transcription and translation of ampicillin resistance
gene.
Some possible products of the transformation
reaction:
Bacterial cell
Genomic DNA
Plasmid w/ insert
Plasmid w/o insert
No plasmid
Ampicillin
resistant
Ampicillin
resistant
No ampicillin
resistance
Nonfunctional
LacZ
Functional LacZ
No LacZ gene
Non circular DNA gets degraded within the cells.
Candidate Identification
• The transformation culture is plated on special media to help identify
which cells have received the recombinant plasmid.
• Two types of media: LB + X-gal, & LB+ X-gal + amp
• Selection:
– Cells with the plasmid can grow on ampicillin media.
– Cells without the plasmid cannot grow on ampicillin media.
• Screening:
– Cells with a functional LacZ gene can convert X-gal to
•
X-gal
– Colorless
------------------------------->
ß-galactosidase
X +
X + gal.
galactose
Blue
• Cells which produce ß-galactosidase form BLUE colonies.
• Cells which are able to grow on ampicillin without ß-galactosidase
production form WHITE colonies. (Suspect  fragment insert)
Some possible products of the transformation
reaction:
Bacterial cell
Genomic DNA
Plasmid w/ insert
Plasmid w/o insert
No plasmid
Ampicillin
resistant
Ampicillin resistant
No ampicillin
resistance
Nonfunctional
LacZ
White colony on
LB+X-gal+amp.
Functional LacZ
Blue colony on
LB+X-gal+amp.
No LacZ gene
No growth on
Ampicillin
Isolating plasmid DNA
• Transformed cells are grown in LB + ampicillin to amplify the target
DNA
• Selective pressure is important.
– E. coli has no instructions for passing the plasmid to the next
generation during cell division.
– The high number of plasmids within each cell slows growth and
lowers the ability to compete.
• Plasmid DNA is amplified in two ways:
– Cell division (Cells multiply in culture)
– High copy replication (multiple copies per cell)
• A plasmid miniprep will be performed on cell cultures to extract and
purify plasmid DNA.
– Cells are disrupted chemically, and the plasmid DNA is separated from
genomic DNA and cellular debris.
Final restriction digest
• Plasmid DNA isolated from transformed cells will be digested with
Hind III, and compared to a known  • Hind III marker.
• Digests will be analyzed with gel electrophoresis for identification of
cloned inserts.
UncutBlue CutBlue UncutWhite CutWhite /Hind III
/ BstE II
Transformation success
• Frequency of transformation = # Transformed cells
Total # of cells in the culture
• Transformation efficiency =
# Transformed cells
Amount of DNA in g