After culturing the bacteria that contain our targeted plasmid, it is time to isolate the plasmid. This an important step because not performing it right will cause in the loss of plasmid. The plasmid DNA is isolated from the cultured bacteria following the alkaline lysis method. The cell is broken down using restriction enzymes in order to isolate the plasmid. In this traditional method, 5 different reagents are used for purification of plasmid. Each reagent performs a special task which some are mentioned as the followings:
50 mM Tris-HCl pH 8.0
10 mM EDTA:
Weakens the cell membrane. It also "limits DNA degradation by binding (chelating) Mg++ ions that are a needed cofactor for bacterial nucleases."
100 ÃÂ¼g/ml RNaseA
1% sodium dodec y l sulfate (SDS):
A 12 carbon chain attached to a sulfate group. Because of its structure, it is considered an amphphilic meaning it can be both hydrophilic and hydrophobic.
It creates holes in the cell memberane in order to break down the cell memberane.
200 mM NaOH:
Denatures both the chromosomal and plasmid DNA into single strands.
Buffer N3: It is a neutralization buffer
4.2 M Gu-HCl
0.9 M potassium acetate:
Causes the SDS from the previous reagent with lipids and protein of the cell membrane to precipitate.
10 mM Tris-HCl pH 7.5
Causes the nucleic acids to precipitate.
10 mM TrisÃÂ·Cl, pH 8.5:
It buffers the solution
Each of these reagents is added in a special time that is mentioned in the method part. The final result is a solution, which only contains the plasmid DNA, and small fragments of chromosomal DNA.
The final product of this step of the project should be about 50 ml of isolated plasmid in a...