Plasmid Isolation from Bacteria

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Purpose

This protocol is used to isolate a desired plasmid from a bacteria containing the plasmid.

Materials Needed

-1.5 mL Microfuge tubes

-Microcolumns with 2 mL collection tubes

-distilled water

-Cell Lysis Buffer

-Neutralization Solution (NSC)

-Endotoxin Removal Wash

-Column Wash Solution

-Micropipette

-Centrifuge

-Bacteria with plasmid DNA

-Broth for bacteria to grow in

Protocol

-Grow the bacteria to saturation in an appropriate broth media overnight

-Centrifuge the bacterial sample in a microfuge tube at 15,000 rpm for 30 seconds

-Dispose of the supernatant and resuspend the pellet in 600 microliters of distilled water

-Add 100 microliters of Cell Lysis Buffer and mix by shaking

-Add 350 microliters of NSC and mix by shaking

-Centrifuge for 3 minutes at 15,000 rpm

-Transfer the supernatant to a microcolumn and centrifuge for 30 seconds at 15,000 rpm

-Discard the flow through and wash the column with 200 microliters of Endotoxin Removal Wash, centrifuge for 30 seconds at 15,000 rpm

-Discard the flow through and wash the column with 400 microliters of Column Wash Solution, centrifuge for 30 seconds at 15,000 rpm

-Discard the flow through and place the column in a fresh microfuge tube

-Wash the column with 30-50 microliters of distilled water, depending on how much bacteria was used, let it sit for one minute, then centrifuge for 30 seconds at 15,000 rpm

-The plasmid is in the water in the microfuge tube

References

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