Team:USTC CHINA/Notebook/Protocols/Extracting plasmids from gram-negative bacterium

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Revision as of 01:12, 26 September 2013

Extracting plasmids from gram-negative bacterium

1.Centrifuge 2-3ml bacteria liquid with 8000rpm and remove the supernatant; resuspense the liquid with lysyme(10mg/ml)~200ul and then leave it at temperature 37℃ for 0.5-1 hour.
2.Centrifuge the liquid with 8000rpm and remove the supernatant; resuspense it with 250uL ddH2O.
3.Add 250uL buffer P2 (Alkaline lysis buffer), make it clear (reverse the tube evenly for several times) and leave it at room temperature for 2-4 minutes.
4.Add 800uL Phenol-chloroform(PH8) and reverse the tube evenly until it layers.
5.Centrifuge the tube for 10 minutes and extract plasmid extraction column from the upper water phase.
6.The following steps are identical with those of Escherichia coli.

Notebook

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