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
E. coli
Lysis solution
Potassium acetate
Isopropanol
RNAse
Loading dye

7. 1. Isolation of Plasmid DNA:
Add 0.4 ml of lysis solution to cells
Gently mix, put on ice for 5 minutes

8. 2. Isolation of Plasmid DNA
Add 0.3 ml of potassium acetate to cell/lyis mixture
Mix gently
Place on ice 4 minutes
Centrifuge (14,000) for 5 minutes

9. 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 5 minutes

10. 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

11. 5. Isolation of Plasmid DNA
Add 50 ul RNase (removes the RNA)
Mix vigorously
Centrifuge 30 sec
Incubate 10 minutes RNase at 37 C
Add 5 ul Loading Dye and store in freezer for next week.

12. Next week
Confirm your isolation with gel electrophoresis