i have performed alkaline lysis method for plamid isolation. 50mM glucose is added in alkline solution I for osmotic balance.
My question is why cant we just bust cell instead of cell lysis for plasmid isolation?
what makes the difference between cell bust and cell lysis
When lysing bacteria for plasmid isolation the protocols are designed to keep as much of the bacterial genome intact and preferably attached to the membrane so that it is removed after lysis. If you used some rough method to break open the cells, you will still get plasmid DNA but are more likely to have much more chromosomal DNA contamination.
When lysing bacteria for plasmid isolation the protocols are designed to keep as much of the bacterial genome intact and preferably attached to the membrane so that it is removed after lysis. If you used some rough method to break open the cells, you will still get plasmid DNA but are more likely to have much more chromosomal DNA contamination.
In the case of bacterial cells, the cytoplasmic soup consists of all the DNA, RNA, proteins and enzymes. The process of cell lysis occurs in a controlled and stepwise manner by compromising the integrity of the cell wall by the combined action of chemicals such as EDTA, SDS, and NaOH. This is then followed by disrupting the H- bonds of DNA by NaOH. However, in this process, the osmotic balance is maintained to avoid rupture of the cell and further mixing of the contents.
Imagine, If you try to burst the cell, without adding any of these chemical reagents, then you are bound to get contamination of chromosomal DNA, RNA, proteins and other cellular debris subsequently affecting your plasmid DNA separation from them.
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