1. Mini-Prep Plasmid Isolation and Identification

2. Page 3-53 in lab manual & handout

3. This is the second experiment (of a total of 3 experiments) for your molecular lab report.

4. This experiment has two components Mini-Prep isolate of plasmid DNA Identification of plasmid DNA by gel electrophoresis (next week)

5. Take cell and gently break them apart Precipitate cellular debris in pellet Save nucleic acids in supernatant Precipitate nucleic acids to a pellet Remove RNA Gel Electrophoresis to confirm isolation

6. Each group pick up the following tubes Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7 Tube 8 E. coli Lysis solution Potassium acetate Isopropanol RNAse Loading dye Cell Resuspension Buffer Plasmid suspension1xTris Buffer

7. 2. Isolation of Plasmid DNA: Add 350 ul of lysis solution to cells Gently mix, put on ice for 5 minutes

8. 1. Centrifuge cells Discard supernatant Resuspend pellet in 200 ul resuspension buffer (tube- 7) “odd group numbers” will also add 5 ul RNAse Incubate room temp. for 5 minutes

9. 2. Isolation of Plasmid DNA Add 200 ul of potassium acetate to cell-lysis mixture Mix gently Place on ice 5 minutes Centrifuge (14,000) for 5 minutes

10. 3. Isolation of Plasmid DNA Carefully remove 0.5 ml of supernatant, place in new tube. This contains your plasmid Add 1.0 ml ice cold isopropanol Place on ice for 10 minutes

11. 4. Isolation of Plasmid DNA Centrifuge for 10 minutes You should now see a pellet of nucleic acids (plasmid DNA + RNA)! Carefully remove & discard all supernatant Air Dry inverted for 10 minutes

12. 5. Isolation of Plasmid DNA Add 60 ul of T.E. buffer to dissolve pellet Remove 40 ul of this dissolved solution and add 5 ul of loading dye To the remaining 20 ul of sample add 20 ul of T.E. Buffer and 5 ul of loading dye. Store in freezer for next week.

13. Next week Confirm your isolation with gel electrophoresis