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
Reconstitute pellet in either RNase or water*
37C min. (longer the better)
Gel Electrophoresis to confirm isolation
*to be further discussed

6. Each group pick up the following tubes
E. coli
Cell Resuspension Buffer
Lysis solution
Potassium acetate
Isopropanol
Plasmid suspension1xTris Buffer
RNAse or water
Loading dye

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)
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)!
Odd number groups add 5 ul RNase and 5 ul Even number groups add distilled water
Carefully remove & discard all supernatant
Air Dry inverted for 10 minutes

12. 5. Isolation of Plasmid DNA
Add 60 ul of 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 Buffer and 5 ul of loading dye.
Store in freezer for next week.

13. Next week
Confirm your isolation with gel electrophoresis