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Biotechnology and Bacterial Transformation

Biotechnology and Bacterial Transformation. ABE Lab sequence. Creating a Recombinant Plasmid. Restriction and Ligation. We used restriction enzymes to cut the two plasmids at the certain points

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Biotechnology and Bacterial Transformation

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  1. Biotechnology and Bacterial Transformation ABE Lab sequence

  2. Creating a Recombinant Plasmid

  3. Restriction and Ligation • We used restriction enzymes to cut the two plasmids at the certain points • The LIG tube contained ligase and ligation buffer. The buffer also had the ATP necessary to help create the bonds • How do we confirm that we got ligation of the plasmid? • What traits do we want in the new plasmid?

  4. Reading Your Gel

  5. The p-ARA-R plasmid

  6. Steps of Bacterial Transformation 1. Competency:Preparing the bacterial cells to receive DNA. • Place bacteria into CaCl2 = CaCl2weakens pores in cell wall. • Rack = breaks up clumps • Cold = prepares Bacteria for next step, concerning opening and closing the membrane.

  7. 2. Transformation: Insert the new plasmid DNA via the pores. • Finger flick = makes DNA touch ALL bacterial cells • Heat Shock = opens pores in the membrane suddenly and pulls in DNA. • Exactly 45 secs. is needed to get DNA deep in Bacteria • Cold = closes pores. Keeps some new DNA in bacteria.

  8. 3. Growth:Resting and feeding stage. • LB Broth (750μL = just enough food for overnight, if you have too little food, bacteria would not be able to grow and reproduce.) • 37oC (Body Temp.) is the perfect temp for bacteria to grow and reproduce

  9. 4. Isolation:Separate the few transformed cells from the entire population. • Ampicillin inhibits growth of bacteria (does not kill) by breaking down cell walls and not allowing bacteria to reproduce. • Only amp resistant bacteria will be able to reproduce. • Satellite colonies form when a bacterium degrades the amp around the cell, enabling other bacteria to reproduce.

  10. 5. Expression: Phenotypic expression. • Arabinose is needed to turn on the rfp gene. • Think about what you know, what type of operon is this? • Expect that all the E.coli that can grow in the presence of ampicillin has been transformed. How do we check to make sure?

  11. Expected results • LB agar plate • LB/ Ampicillin • LB/ AMP/ARA - You need to record number of colonies and color for both +DNA and -DNA plates

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