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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

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molecular cloning

Molecular Cloning

Biology 20L

Spring 2003

overview of molecular cloning
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 cloning3
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
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.
slide7

•BstEII pUC19 pUC19 •HindIII  •HindIII •BstEII

puc19 a genetically engineered plasmid
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.
slide9

http://www.fermentas.com/techinfo/NucleicAcids/mappuc1819.htmhttp://www.fermentas.com/techinfo/NucleicAcids/mappuc1819.htm

slide10

T4 Phage

T4 phage infecting a bacterial cell

ligation reaction
Ligation Reaction
  • Hind III breaks covalent bonds at the recognition sequence.
    • A•AGCTT A AGCTT
    • T TCGA•A TTCGA 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
Some possible ligation reaction products:

Recombinant No insert Fragments No ligation

transformation
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
Some possible products of the transformation reaction:

Bacterial cell

Genomic DNA

Plasmid w/ insert

Ampicillin resistant

Nonfunctional LacZ

Plasmid w/o insert

Ampicillin resistant

Functional LacZ

No plasmid

No ampicillin resistance

No LacZ gene

Non circular DNA gets degraded within the cells.

candidate identification
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.
  • X-gal -------------------------------> X + galactose
    • Colorless ß-galactosidaseBlue
  • 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)
slide16

Some possible products of the transformation reaction:

Bacterial cell

Genomic DNA

Plasmid w/ insert

Ampicillin resistant

Nonfunctional LacZ

White colony on LB+X-gal+amp.

Plasmid w/o insert

Ampicillin resistant

Functional LacZ

Blue colony on LB+X-gal+amp.

No plasmid

No ampicillin resistance

No LacZ gene

No growth on Ampicillin

isolating plasmid dna
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
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.
transformation success
Transformation success
  • Frequency of transformation = # Transformed cells Total # of cells in the culture
  • Transformation efficiency = # Transformed cells Amount of DNA in g