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 E. coli Lysis solution Potassium acetate Isopropanol RNAse Loading dye

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