STEP 4: Deproteination: 

8. Resulting supernatant containing plasmid DNA and protein is treated with equal volume of phenol: chloroform: isoamyl alchol mixture and mix by vortexing for 1mins. This step removes protein from the solution in the precipitate where as plasmid remained in solution.
9. Centrifuge at 12000g for 5mins and transfer the upper clear supernatant to fresh tube.
10. Add equal volume of chloroform:isoamyl (24:1) and mix by vortexing.
11. Centrifuge at 12000g for 5mins and transfer the upper clear supernatant to fresh tube.

STEP 5: Precipitation :  

12. Add 2 volume of absolute alchol and mix by vortexing to precipitate the plasmid DNA.
13. Centrifuge at 12000g for 5mins and discard the supernatant. Wash the DNA pellet with the 70% ethanol.
14. Centrifuge at 12000g for 5mins and discard the supernatant.
15. Air dry the pellet and dissolve the pellet in the sterile molecular biology grade water or TE Buffer (Tris pH 8.0 containing 10mM EDTA).  
16. Analyze the DNA on the 0.8% agarose gel.
Observation: Analysis of plasmid DNA on the agarose gel gives 3 bands corresponding to the 3 different forms of DNA (Figure 39.3).

Figure 39.3: Analysis of plasmid analyzed on the agarose gel.