Approximately 1.5 ml of the overnight culture was placed in eppi tubes. The eppi tubes and the contents were centrifuged for 5 min at 10,000 Ã— g. The supernatant was removed and discarded. The pellet was resuspended in a cell resuspension solution (250Î¼l). 250Î¼l Cell Lysis Solution was then added, and the contents mixed by inverting tubes 4 times, followed by incubation for 5 minutes. Alkaline protease solution (10Î¼l) was added and mixing accomplished by inverting tubes 4 times, followed by incubation at room temperature for 5 minutes. 350Î¼l Neutralization Solution was added and mixing accomplished by inverting tubes 4 times.
A maximum speed of 14,000 Ã— g speed was used to centrifuge the bacterial lysate in microcentrifuge at room temperature, for 10 minutes. A decantation process was then used to transfer the cleared lysate to the already prepared spin column. The supernatant was transferred into a microcentrifuge and centrifuged at maximum speed at room temperature, for 1 minute. To the spin column was added 750Î¼l Column Wash Solution. The content was placed in a microcentrifuge and centrifuged at room temperature for I minute at maximum speed. The spin column was then removed from the tube and the flow through discarded.
Afterwards, the spin column was reinserted into the collection tube. The wash procedure was repeated using 250Î¼l of Column Wash Solution. The contents were transferred into a microcentrifuge and centrifuged at room temperature for 2 minutes, at maximum speed. The spin column was transferred to a new, sterile, microcentrifuge tube. The plasmid DNA was eluted by an addition to the spin column of 100Î¼l of Nuclease-Free Water. The contents were transferred to a microcentrifuge and centrifuged at room temperature for 1 minute at maximum speed
Once the DNA had been eluted, the assembly was removed from the 1.5 ml microcentrifuge. The spin column was tubed and discarded. When stored at or below â€“20Â°C, DNA has the ability to remain stable in water without the need to add a buffer. The sample was then run on the gel agarroass
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