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Transformation Procedure Summary Incorporation of Foreign DNA Adhere DNA to Cells on ice Heat shock and DNA Entry Recove

Transformation Procedure Summary Incorporation of Foreign DNA Adhere DNA to Cells on ice Heat shock and DNA Entry Recovery on ice Plating Cells Screen for Transformants. 10 ul of pGlo (loopful of plasmid DNA) + one colony. 250 ul CaCl 2 (Transformation Solution) on ice for 10 min.

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Transformation Procedure Summary Incorporation of Foreign DNA Adhere DNA to Cells on ice Heat shock and DNA Entry Recove

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  1. Transformation ProcedureSummary Incorporation of Foreign DNA Adhere DNA to Cells on ice Heat shock and DNA Entry Recovery on icePlating Cells Screen for Transformants 10 ul of pGlo (loopful of plasmid DNA) + one colony 250 ul CaCl2 (Transformation Solution) on ice for 10 min 42°C for 50 sec Be exact! Place cells on ice 2 min. Take out and add 250 ul of RT LB  Recover for 10 min Plate 100 ul per plate Spread, 37°C O.N.

  2. Typical Results 112 117 Clear

  3. Calculating transformation efficiency Transformation efficiency = # of cells transformed/ug of DNA Stock [pGLO] = 20ug of DNA in 250ul of buffer and we used 10 ul per transformation Total volume of suspension = volume of CaCl2 + volume of pGLO added + volume of L.B. added 100 ul of the suspension put onto each plate

  4. What do I hand in? • Three questions: • What was the transformation efficiency? Show calculation. • Why did each plate (-/+pBlu) turn out the way it did? • What might have happened in our experiment?

  5. Engineering your gene of choice  Called Recombinant DNA Gene Gene

  6. Practically speaking, what’s the purpose of all of this?

  7. Applications: • Detecting infectious diseases • Diagnosing genetic disorders • Human gene therapy • Production of important proteins • such as insulin and growth hormone • 5. Forensic applications • 6. Transgenic plants • 7. Environmental applications

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