How do I isolate plasmid DNA from E coli?
Posted September 28, 2020
Plasmid DNA isolation has to be performed carefully to avoid genomic DNA contamination. Alkaline lysis has been the most preferred method to achieve it. The following steps can be taken to isolate plasmid DNA from E. coli.
- E. coli cell pellet is resuspended in suspension buffer containing tris, EDTA, glucose and RNase A.
- Lysis buffer containing NaOH and SDS is added. The lysis buffer would completely denature plasmid and genomic DNA.
- Neutralization buffer containing potassium acetate is added to neutralize the sample. During this step, plasmid DNA would renature into dsDNA as it is small. However, genomic is too long to reanneal fully.
- Centrifugation step would pellet genomic DNA along with other cellular debris and plasmid DNA remains in the supernatant.
- The plasmid DNA present in the supernatant could be precipitated with ethanol, followed by usually followed by a “wash” step using 70 % ethanol.
- At last, resuspend the purified DNA in water for easy handling and storage.
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