pARA -R Restriction Digest: An Introduction to Plasmids and Restriction Enzymes

1. pARA-R Restriction Digest:An Introduction to Plasmids and Restriction Enzymes Laboratory 2a

2. Overview • Purpose: • Examine role of restriction endonucleses (enzymes) in genetic engineering • Examine a bacterial plasmid and its use in biotechnology • Methods: • Preparing the pARA – R restriction digest

3. Introduction • Restriction enzymes • Cut DNA molecules from various organisms and recombine pieces • Recombinant DNA • Restrict the growth of viruses in bacteria • Digest the DNA molecule at specific nucleotide sequences • Restriction fragments • DNA fragments • Sticky ends • Allow annealing and recombination of DNA fragments from different sources

4. 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

5. Introduction • Bacterial plasmids • Circular pieces nonessential DNA found in bacteria • Can be engineered to carry genes • Express proteins encoded by these genes • pARA – R • Recombinant DNA plasmid • Engineered to express rfp gene • Produces a mutant Red Fluorescent Protein (mFP)

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

8. Recombinant Plasmid pARA - R • Important control elements: • araC • Protein to help bacteria make other proteins • Encoded by genes inserted into plasmid • pBAD • Site where RNA polymerase binds to initiate transcription • rfp • Hind III & BamH I • Restriction enzymes • ampr • Antibiotic resistance gene • Encodes beta lactamase

9. Materials • Reagents: • pARA-R (70 ng / μL) • Restriction enzymes • BamH I • Hind III • 2.5x restriction buffer • Distilled water (dH2O) • Equipment & Supplies • P-20 micropipette and tips • 1.5 mL microfuge tubes • Minicentrifuge • 37oC water bath • Markers

10. What will you need to do? • Aliquot: • pARA – R • Enzyme mix • 2.5x restriction buffer • Turn on water bath the day before lab • 37oC

11. Methods

12. 1. Preparing the pARA-R Restriction Digest • Three tubes: • pARA-R • Enzyme mix • 2.5x restriction buffer • Obtain 2 clean 1.5 mL microfuge tubes • Label as “A+” and “A-” • Use fresh tip and add 4 μL of 2.5x restriction buffer to both tubes • Add 2 μL dH2O to “A-” • Fresh tip and add 4 μL of pARA – R to both tubes Double these numbers!! = 20μL

13. 1. Preparing the pARA-R Restriction Digest • A+ tube • Teacher will dispense enzyme mix • 2 μL • Cap tubes and gently “flick” to mix • Minifuge • Balance with other tubes • Place both tubes in 37oC water bath for 60 minutes • Either go directly to Lab 4a or freeze until ready to complete Double these numbers!! = 20μL

14. Conclusions • Will result in 2 restriction fragments • 4018 bp with ampr gene and control regions • 702 bp with rfp gene

15. Restriction analysis of pARA-R Bruce Wallace Restriction fragments after digest with Hind III and BamH I BamH I Hind III 4018 bp BamH I Hind III 702bp