Mini-Prep Plasmid Isolation and Identification

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

Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 E. coli Lysis solution Potassium acetate Isopropanol RNAse Loading dye Each group pick up the following tubes

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

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

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

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 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.

Next week • Confirm your isolation with gel electrophoresis

Mini-Prep Plasmid Isolation and Identification