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

General Overview
(some of these steps have been completed for you*
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 15-30 min. (longer the better)

Gel Electrophoresis to confirm isolation

*to be further discussed

Each group pick up the following tubes
Tube 1 E. coli for DNA extraction
Tube 2
Potassium acetate
Tube 3
Isopropanol
Tube 4A
Tube 4B RNAse
Tube 5 DI water
Dilution buffer

-----------------------------------------
Loading dye

Tube 8
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

Re-suspend pellet in 200 ul re-suspension buffer

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

Odd number groups now add 5 ul RNase and 5 ul Even number

groups add distilled water
5. Dilution of Plasmid DNA

Add 60 ul of buffer to previously dissolve pellet

Remove 20 ul of this dissolved solution and add 5 ul of loading dye

To the remaining 20 ul of sample +10 ul buffer and 10 ul of loading

dye.

Run gel!!
Next week
Confirm your isolation
with gel electrophoresis