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Overview of Cloning a DNA Fragment into a Plasmid Vector

Overview of Cloning a DNA Fragment into a Plasmid Vector. insert. Bacterial Transformation = Introduction of plasmid DNA into E. coli. Requires “competent” E. coli (treated w/ CaCl 2 ). Plasmids are Extrachromosomal Self-Replicating DNA molecules. Features of plasmids :. High yields of

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Overview of Cloning a DNA Fragment into a Plasmid Vector

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  1. Overview of Cloning a DNA Fragment into a Plasmid Vector insert Bacterial Transformation = Introduction of plasmid DNA into E. coli Requires “competent” E. coli (treated w/ CaCl2)

  2. Plasmids are Extrachromosomal Self-Replicating DNA molecules Features of plasmids: High yields of plasmid DNA 1) Replication origin (ORI)—allows the plasmid to be replicated by the host (usually a “high copy number” ORI 500 plasmids/bacterium) 2) Selectable marker(s)—such as the ampicillin resistance gene permits for selection of the plasmid 3) Cloning site(s)—allows for the insertion of exogeneous DNA Q: What Makes the Insertion of DNA into Plasmid Vectors Possible?

  3. A: Restriction Endonucleases (enzymes that cleave DNA at specific sites) The Restriction-Modification System of Bacteria Foreign DNA Host DNA Part II: Modification Part I: Restriction Bacteria produce restriction enzymes that digest foreign (viral DNA) Bacteria methylate their DNA to protect it from digestion

  4. There are Three Types of Restriction Endonucleases Type II Restriction Enzymes are the Most Useful for Cloning 1) They recognize palindromic sequences They make double-stranded cuts 2) They cut at the site of recognition They generate predictable ends

  5. Examples of Type II Restriction Endonucleases Blunt ends Cohesive “sticky” ends

  6. Q: How Frequently Will a Restriction Enzyme Cut DNA? A: It Depends of the Length of the Recognition Sequence A four-base cutter: (Sau3A) GATC 1/4 X 1/4 X 1/4 X 1/4 = 1/256 bp A six-base cutter: (BamHI) GGATCC (1/4)6= 1/4096 bp

  7. Agarose Gel Analysis of Digested DNA Fragments Two Types of Gels Used for Separating DNA Fragments Agarose Gels: very porous; ideal for large fragments (0.5-20 kb) Polyacrylamide Gels: small pores; ideal for small fragments (0.1-1 kb)

  8. EcoR I + Sma I MW Uncut EcoR I Sma I 6 kb/0 5 1 6 Kb 5 4 4 2 3 3 2 1 Restriction Mapping Problem #1 3X (3, 2, & 1 Kb) 2X (4 & 2 Kb) 1X (6 Kb) EcoR I Sma I Sma I 1) How big is the plasmid? 2) How many times was the plasmid cut? What are the sizes of each band? 3) Do the band sizes add up the total plasmid length? (Are the fragments in equal stochiometric amounts?) 4) In double digests, which band(s) gets cut by the other enzyme?

  9. Restriction Mapping Problem #2 2X (5&1) 2X (4&2) 2X (3) 1X (6 Kb) 1X (6) 1X (6) Hind III -Pst I Hind III -BamH I Pst I -BamH I Hind III MW BamH I Uncut Hind III Pst I 6 kb/0 6 Kb 5 5 1 4 3 2 4 2 1 3 1) How big is the plasmid? 2) How many times was the plasmid cut? What are the sizes of each band? 3) Do the band sizes add up the total plasmid length? (Are the fragments in equal stochiometric amounts?) 4) In double digests, which band(s) gets cut by the other enzyme?

  10. An Example of a Sticky End Ligation *DNA ligase can also religate (reseal) a cut plasmid (ligate without the insert)!

  11. DNA Ligase • Catalyzes the formation of a phosphodiester bond between a 3’ hydroxyl • at the end of one DNA strand and the 5’ phosphate at the end of another strand.

  12. IV. Cloning Vectors A) Bacterial Plasmids An Expression-Cloning Vector

  13. Gene X An Expression Vector for Making a LacZ Fusion Protein Ligate gene of interest into any of these sites Gene of interest must be ligated “in frame” with lacZ coding sequence Lac Z—Protein X N C LacZ can be replaced with: Affinity Ligand: 1) Glutathione S-transferase (GST) Glutathione 2) Maltose-binding protein (MBP) Starch (amylose)

  14. Limitations of Plasmids: 1) Can ligate large inserts into them (<10 Kb inserts) 2) Low efficiency of transformation 3) Large colony size  Can’t screen manypotential recombinants per plate (~5,000 colonies /plate) Bacteriophage Lambda ()—Very Useful for Cloning Large Inserts (25 kb) Lambda Virus Infection Results in Plaques

  15. How to Make a Genomic Library Using l Virus cos sequences at ends of lambda genome are neededfor packaging the DNA into empty virus heads cos cos Thus only recombinants with 20-25 kb inserts will produce plaques

  16. Advantages of l virus vectors: 1) Accepts large inserts (25 Kb) 2) Efficient infection (compared to low transformation efficiency of plasmids) 3) Produce tiny plaques Can screen (~50,000 plaques/plate) Disadvantages of l virus vectors: 1) Not as easy as working with plasmids

  17. Scenario: You want to screen a library for a particular gene insert and 1) Determine the coding sequence of the insert or 2) You want to express the gene in E. coli Problem: Genomic libraries (from eukaryotes) have introns Solution: Remove the introns by cloning mRNAs instead of genomic DNA

  18. V. cDNA Libraries (converting mRNA into “complementary DNA”

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