Material and Instrument:

1. E. coli cells culture (~1.5ml) containing plasmid of interest. 
2. Solution I (50 mM glucose, 25 mM TrisHCl pH 8.0, 10 mM EDTA pH 8.0): Prepare the solution of 50 mM TrisHCl pH 8.0, 50 mM EDTA pH 8.0 and sterile by autoclave. Prepare the 100mM Glucose and sterile by filter through 0.2µm. Solution-I is prepared by mixing both solution to prepare solution-I.  
3. Solution II (0.2 N NaOH and 1% SDS): This solution has to prepare fresh by mixing stock solution of 2N NaOH and 10% SDS in sterile molecular biolog grade water. Don’t vortex this solution and discard any left-over solution.
4. Solution III (potassium acetate, glacial acetic acid.):
5. Phenol : Chloroform: Isoamyl alchol: Mix Phenol (equliberated at pH 8.0), chloroform and Isoamyl alchol in the ratio of 25:24:1,mix by vortexing.
6. Absolute Ethanol:
7. Luria Bertani medium: The composition of the luria Bertani and other bacterial expression media is given in Table 37.2. For preparation of media dissolve the components in 1 liter of distilled water. Cover the top of the flask with cotton plug or aluminium foil and autoclave the solution at 121°C for 20 minutes. The various antibiotics or nutrient supplement should be  added to the media when the temperature is less than 50°C (Figure 37.1 ).
8. Autoclaved molecular biology grade water 
9. Agarose and Buffers for Electrophoresis

Methods: The different steps in isolation in bacterial plasmid is given in Figure 39.2.

STEP 1 (resuspension and lysis of bacterial cells): The bacteria containing plasmid was grown in suitable culture media in high density (~0.8 optical density). Each Bacterial cell contains chromosomal DNA, plasmid DNA and cellular proteins.
1. 1.5 ml bacterial culture is collected by centrifugation at the bottom and discard the supernatant and invert the tube on a dry paper towel to dry the pellet.  
2. Resuspended the bacterial pellet in 0.1 ml solution I containing 50 mM glucose, 25 mM TrisHCl pH 8.0, 10 mM EDTA pH 8.0. Break the cell pellet by vortexing for 2mins.
3. Incubate the  Bacterial pellet on ice for 5mins. This step will partially lyse the bacteria by hypotonic osmosis and releases cellular content.

STEP 2 (Alkaline Lysis):

4. Bacterial cells are treated with 0.2ml solution II containing 0.2 N NaOH and 1% SDS. Don’t vortex but mix by inversion. Incubate the tube on ice for 5mins. This step will completely lyse the cells and denature DNA (both chromosomal and plasmid DNA) and protein.