1. Bacterial Transformation The Cohen - Boyer Experiment

2. Objective: To introduce antibiotic resistant plasmids into the genes of E. coli.

3. Procedure 1)Add restriction enzyme EcoRI to the DNA samples - cuts between base pairs and creates “sticky ends”

4. Procedure (cont.) 2) Mix the samples (put resistant gene into plasmid) 3) Add ligase - repairs the cut site/recombines the fragments

5. Procedure (cont.) 4) Insert plasmids (with and without new gene) into E. coli cells 5) Heat shock the E. coli - opens cell membrane to introduce the new plasmid into the cells and changes shape of DNA 6) Isolate the plasmids containing the gene by nucleic acid hybridization with a nucleic acid probe - hydrogen bonds specifically to complimentary strands of the gene by labelling it with a radioactive isotope or fluorescent tag

6. Differentiating the Cells The bacterial colonies with the original plasmids will appear white or gray The bacterial colonies with recombinant plasmids will appear yellow or blue

7. More Info Also summarized in this slide that you should have read in the notes

8. FUTURE LAB Inserting glow gene into bacteria !!!!