The Various Steps of the Alkaline Lysis Mini-Prep Procedure

This describes the steps involved in the process of Alkaline lysis mini-prep procedure.

1. Centrifuge - this original centrifugation step is required to harvest the bacterial cells ie to separate them from the liquid growth medium which is discarded as the supernatant.

2.Re-suspend Pellet in solution 1- Solution 1 contains glucose, Tris-Cl and EDTA and the pellet is resuspended in this solution to chelate divalent metals (through the action of EDTA) which destabilizes the bacterial cell membrane and inhibits the action of DNases which would destroy the DNA that is wanted. The glucose is added to maintain the osmolarity and prevent the bursting of the cells by the buffer.

3. Add solution 2 and mix - Solution 2 contains NaOH and 1% sodium dodecyl sulphate (SDS) making it a highly alkaline solution. The SDS creates holes in the cell membranes and NaOH loosens the cell walls and releases the DNA. NaOH also denatures the chromosomal DNA through linearization and eventual separation while the plasmid DNA remains the same due to its topological constraints.

4. Add solution 3 and mix - Solution 3 contains Potassium acetate, glacial acetic acid and distilled water. The KAc allows circular DNA to renature while cellular DNA remains denatured as ssDNA, which is then precipitated due to its insolubility in high salt solutions.

5. Add LiCl, mix and centrifuge - the salt lithium chloride is added to precipitate the RNA which can then be easily discarded.

6. Remove supernatant, add isopropanol to supernatant and mix - the reagents at this step facilitates the process of alcohol precipitation.

7. Centrifuge and discard supernatant - centrifugation at this stage allows the plasmid DNA to form a pellet while all of the waste materials and contaminants remain in the supernatant and are discarded.

8. Add ethanol, centrifuge, discard supernatant then dry - the ethanol is added at this stage to remove any contaminating salts since the ethanol retains the DNA as a precipitate while contaminating salts dissolve in solution and can be removed. This is allowed to dry to ensure the evaporation of the ethanol.

9. Resuspend pellet in TE buffer, add RNase - this step is important in the removal of any RNA contaminants through the action of the RNase, which functions to break down RNA into its nucleotide components. TE buffer is used to store DNA as the EDTA in TE chelates Mg²⁺ and other divalent metals ions needed for most DNA degradation reactions and hence suppresses it.

References
1. Becker J. Caldwell G. "Biotechnology, A Laborator Course" Academic Press, 1996.