1. Lab Exam 2 Overview

2. Bacterial Transformation To impart new phenotype by adding expressible genes Why use bacteria? – Rapid growth – Plasmids as vectors – Relatively less complex gene expression

3. Prepare Recombinant Plasmids Prepare Competent Cells Transform Competent Cells Select for Transformed Cells Confirm Transformation By Plasmid Isolation and Analysis

4. Prepare Recombinant Plasmids Structure of a plasmid – Must contain an antibiotic resistance gene Digest plasmid and foreign DNA with same restriction enzyme – Sticky ends Mix digested DNAs and combine with ligase – Recombinant contains plasmid DNA ligated with foreign DNA

5. Our Recombinant Plasmids

6. Prepare Competent Cells Incubation in CaCl 2 solution – Opens ‘pores’ in plasma membrane – Helps plasmid DNA adhere to bacterial cells

7. Transformation of Competent Cells Mix ligated DNAs with competent cells Heat shock to draw plasmid into cells Recovery in LB Plating on LB/agar – LB – LB/amp – LB/amp plus

8. Selection for Transformed Cells New phenotypes due to presence of plasmid in transformed cells and expression of genes in plasmid DNA Growth in presence of ampicillin due to expression of ampicillin resistance gene (β- lactamase) Expression of other genes in recombinant plasmid, e.g. GFP, lacZ – GFP – green glow in UV light – lacZ – Blue/white selection

9. New Phenotypes pGLO Transformed CellspUC/λDNA transformed cells Blue/white Selection Ampicillin Resistance

10. Confirmation of Transformation by Plasmid Isolation and Analysis Expand population of TX cells Isolate plasmid Digest isolated plasmids to test for presence of foreign DNA Analyze digested plasmid by agarose gel electrophoresis

11. Confirmation of Recombinant Plasmid