1. Ch. 2A: How Do You Begin to Clone a Gene?

2. Learning goals Describe the characteristics of plasmids Explain how plasmids are used in cloning a gene Describe the function of restriction enzymes Explain how to use restriction enzymes to create a recombinant plasmid

3. Key Ideas Plasmids - ideal vectors for genetic engineering ◦ replicate in the bacteria cell ◦ gene promoter ◦ antibiotic resistance as a selectable marker, ◦ can be transferred into bacteria by conjugation. Restriction enzymes – key to the creation of a recombinant plasmid. ◦ cut DNA at specific sequences ◦ sticky ends allow strands to join

4. The Plasmid pARA-R plasmidplasmid

5. Clone That Gene activity 1. Cut the plasmid and the human DNA with the appropriate restriction enzyme 2. Insert the insulin gene into the plasmid DNA 3. Determine which antibiotic you would use to identify bacteria that have taken in the plasmid

6. Tips Reagents should be stored in a freezer until you are ready to prepare them for students. Allow to defrost for 15 minutes before using. The reagents can be aliquoted up to several days before the lab, then store in the freezer/refrigeratoraliquoted Vortex and spin enzyme mix and 2.5x RB before aliquoting video

7. Tips Calibrate and mark the controller “Floatie” marked with team number. Each period has a different color. Bottom of tubes in the water Low-tech water bath Multiple pans allow tubes from different classes to stay separated

8. BamH I Hind III BamH I Hind III Restriction digest of pARA-R Recombinant plasmid of interest pARA-R 5,302 bp Biotech Experience P BAD -rfp 806 bp

9. Restriction analysis of pARA-R Restriction fragments after digest with Hind III and BamH I Biotech Experience 806 bp BamH IHind III BamH IHind III 4,496 bp

10. Go to Student Guide page 46 and complete Lab 2A - Preparing to Verify the RFP Gene: Digesting the pARA-R Plasmid