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Posted

Does anyone know of a simple procedure for isolating DNA from E. coli?

I have considered using a freeze-thaw method  with a freezer to lyse the cells, but I don't know if that would be most efficient. I have also seen some methods that where phenol is simply used to do it. If I did use the freeze-thaw method, would I need a buffer in the solution? How much bacteria should I put in the water to be freezed?

For the DNA isolation, I wanted to use a centrifuge with equal parts of the cell mixture and phenol, but I'm not sure how long I would need to do that for. Is that the correct mixture to put in the centrifuge? After centrifugation, how would I separate the phase containing the DNA?

Thank you!

Posted

This is just a rough overview of the DNA isolation of E. coli:
Centrifuge
Discard supernatant
Add lysis buffer
Add phenol
Spin 
A white layer should appear at the bottom and an aqueous upper layer and a middle phenol layer
Remove the upper aqueous layer and add to tube
To remove phenol add equal volume chloroform 
Spin and remove aqueous layer to new tube
Now you have to precipitate the Dna
Add ethanol to precipitate Dna
Spin
See the Dna pellet and discard the supernatant
Rinse in 70% ethanol
Resuspend in TE buffer (Add RNase to digest RNA)
Check on agrose gell

(https://bio-protocol.org/bio101/e97)

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