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Laboratory: Bacterial Transformation

Laboratory: Bacterial Transformation. Introduction of plasmid DNA into E. coli. This laboratory is. The first part in a series of 3 experiments: Plasmid Transformation Plasmid Isolation Plasmid Mapping. Transformation. A process of plasmid DNA uptake

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Laboratory: Bacterial Transformation

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  1. Laboratory:Bacterial Transformation Introduction of plasmid DNA into E. coli

  2. This laboratory is • The first part in a series of 3 experiments: • Plasmid Transformation • Plasmid Isolation • Plasmid Mapping

  3. Transformation • A process of plasmid DNA uptake • In our experiment the plasmid is: extrachromosomal

  4. Transformation experiment illustrates: • Genotype determines phenotype

  5. Plasmid DNA How will the phenotype of the E. coli be changed?

  6. Plasmids have selectable markers to detect change: • Color alteration of colonies • Antibiotic resistance

  7. Let’s look more closely at “our” plasmid Amp r pGal Lac Z gene

  8. What are characteristics of the lac Z gene?

  9. Lac Z gene • Codes for beta-galactosidase • Beta-galactosidase is secreted by the transformed E. coli • Beta-galactosidase utilizes the substrate “X-gal” to produce a blue color

  10. What are characteristics of ampr gene?

  11. Amp resistance gene • Beta-lactamase secreted extracellularly • Beta-lactamase inactivates ampicillin

  12. How to transform cells. • Competent bacterial cells are required • Introduction of plasmid DNA + bacteria • “Heat Shock” to increase uptake of DNA

  13. Bacterial Tranformation • Protocol

  14. Experimental overview: Please refer to your lab manual.

  15. Group materials • Each group • Plasmid DNA • Buffer • Recovery broth • 3 agar plates • 3 transfer pipets or use micropipettors • 2 “yellow platers”

  16. Plating of transformed bacteria Cell spreader Gently spread across surface Let plate sit 10-15 min. Cover Incubate 37 overnight Agar plate with drops of transformed cells

  17. SUMMARY This is in your lab manual! • Incubate 10 min. on ice • Incubate 42 C for 90 seconds • Place on ice for 1 minute • Add 0.75 ml recovery broth to control and treatment tubes • Incubate at 37 C 15-30 min • streak 10 drops of cells evenly Treatment Control Amp/X-Gal X-Gal Amp/Xgal

  18. Next lab: Transformation Efficiency is Determined • # of transformants/ug of DNA x volume at recovery (ml)/volume plated (ml)= • # of transformants per ug of DNA Our experiment uses: DNA concentration: 0.025 ug Recovery Volume: .68 ml Plating Volume: 0.25 ml

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