LucaAster Posted March 2, 2019 Posted March 2, 2019 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!
Vexen Posted March 7, 2019 Posted March 7, 2019 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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