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