Prof. Dr. Sevgi ERTUĞRUL KARATAY
Isolation of plasmid DNA by alkaline lysis method
Plasmids are extrachromosomal DNA structures found in bacteria and some yeasts, which do not encode vital genes, but
which can help the survival of microorganisms (antibiotic resistance, some enzymes, etc.).
Plasmids are very important in molecular biology because they are frequently used in cloning studies.
• The basis of the alkaline lysis method is that the pH changes in the environment have a different effect on chromosomal and plasmid origin DNA fragments.
• Basically, when the medium becomes alkali, both chromosomal and plasmid DNA become a single strand. However, when the pH of the medium is lowered and acidified again, the strands of the small plasmid DNA can merge rapidly, but since the chromosomal DNA is larger, the double-chain formation does not occur easily and the chromosomal DNA is denatured.
• In this way, chromosomal DNA and plasmid DNA can
be separated.
• Procedure
• Requirements
• Overnight bacterial culture (E. coli DH5α)
• Sterile Eppendorf Tube
• Sterile micropipette tip
• Tube holder
• Automatic pipette
• Microfuges