Mini-Prep Plasmid Isolation and Identification
Page 3-53 in lab manual & handout
This is the second experiment (of a total of 3 experiments) for your molecular lab report.
This experiment has two components
Mini-Prep isolate of plasmid DNA
Identification of plasmid DNA by gel electrophoresis (next week)
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
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
2. Isolation of Plasmid DNA:
Add 350 ul of lysis solution to cells
Gently mix, put on ice for 5 minutes
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
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
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
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
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.
Next week
Confirm your isolation with gel electrophoresis