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

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 plasmid

5. pARA-R construct Bruce Wallace Recombinant plasmid of interest BamH I pARA-R 5,302 bp PBAD-rfp 806 bp Hind III

6. BamH I sticky end Hind III sticky end Hind III sticky end BamH I sticky end Engineering the Plasmid: ligation of rfp gene into p-ARA Bruce Wallace 3’ 5’ 3’ 5’ 5’ 3’ 5’ 5’ 3’ 3’ 3’ 5’

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

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

9. Learning goals: lab #4 Describe why it is important to verify products created in the genetic engineering process Predict the relative speed of DNA restriction fragments and plasmids through a gel during gel electrophoresis Separate and identify DNA restriction fragments and plasmids using gel electrophoresis

10. Key Ideas The multistep process that is used to clone a gene results in multiple products • Need to verify that you have the recombinant plasmid you need. DNA fragments and plasmids can be separated by gel electrophoresis. Loading dye helps monitor the progress of the gel electrophoresis procedure. DNA ladder helps determine the sizes of unknown pieces of DNA Gel is stained in order to show the location of the DNA fragments and plasmids.

11. Review questions • Why is it important to have sticky ends? • What is the purpose of the restriction enzymes? • How do you confirm the uptake of the gene into the plasmid?

12. 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 Clone That Gene activity

13. 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/refrigerator Vortex and spin enzyme mix and 2.5x RB before aliquoting video

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