1. Verify recombination by electrophoresis. Digest of rfp gene. Transform bacteria with recombinant plasmid. Recombination (ligation) of plasmid and rfp gene. Induce expression of rfp gene. Observe bacteria. Digest of plasmid

2. Journal 12.02.11 1)Copy the steps on the other slide in the correct order. 2)Flip the pages in the book: which steps correspond to labs 2a, 4a, 5a? 3)At which step is there a process of gene expression? (Can be answered in 3 columns)

3. DO: Lab 2a – Restriction digest 1. Mix reagents, as described in page 2a.3 -Reaction buffer mix -Plasmid -Water (to one test tube) -Add enzyme: from teacher. 2. Place at 37oC for at least 1 hour.

4. PreLab 2a – Q & A Animation: Plasmid digestion Group papers: Lab 1 – Conclusions page 1.6 Lab 2a – Conclusions page 2a.4 -Pour Gel (not in 2013)

5. http://Plasmid RecombinationPlasmid Recombination Gene Cloning Animation

6. pARA-R construct Recombinant plasmid of interest pARA-R BamH I Hind III rfp 702bp 4720 bp

7. rfp – Gene for Red Fluorescent Protein, originally from the Sea Anemone Discosoma sp.

8. To presenting students: The following slide is from the alternative lab sequence, where students also performed the ligation step. The two plasmids: One served as the ‘source’ of the rfp gene, and one as the ‘vector’ which would turn into our pARA-R.

9. Restriction analysis of pKAN-R and pARA Bruce Wallace BamHI HindIII BamHI HindIII pKAN-R 5,512 bp pARA 4,872 bp P BAD -rfp 806 bp 376 bp

10. Restriction analysis of pKAN-R and pARA Restriction fragments after digest with Hind III and BamH I Bruce Wallace 4,706 bp BamH IHind III 806 bp BamH IHind III 376 bp BamH IHind III BamH IHind III 4,496 bp

11. Engineering the Plasmid: ligation of rfp gene into p-ARA sticky end BamH I sticky end Hind III sticky end BamH I sticky end Hind III

12. Restriction digest of pARA-R Recombinant plasmid of interest pARA-R BamH I Hind III rfp 702bp 4720 bp

13. To presenting students: Refer to Lab3 (background and conclusions) in the student guide for more information.Lab3 Key points: -Why 70oC incubation (Denaturation – what does this mean?) - What does ligation have to do with ‘recombinant DNA’?