add 6 ml of bacterial culture in LB medium to a 15 or 50 ml conical tube. Alternatively, centrifuge up to 12ml, (or for low-copy number plasmids, 24ml) of bacterial culture in a 50 ml conical tube for 10 minutes at ≥3,400 x g. Discard the supernatant
add 6 ml of TE or water to the bacterial cell pellet and completely re-suspend by vortexing or pipetting. IMPORTANT: If culture is grown in a medium other than LB or contains a high-copy number plasmid, the culture volume processed must not exceed 12 ml. Processing an excess of bacteria will reduce lysis efficiency resulting in lower yields of plasmid DNA
add 1 ml of 7X Lysis Buffer (Blue) to the sample and mix by gently inverting the tube 4-6 times. Let sit at room temperature for 5 minutes
add 3.5 ml of cold Neutralization Buffer (Yellow) and invert 4-6 times to mix thoroughly. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete neutralization
place the Zymo-Midi Filter™/Zymo-Spin™ V-E column assembly into a clean 50 ml conical tube
add the entire mixture into the blue Zymo-Midi Filter™ column, place the cap on the conical tube, and centrifuge at 500 x g for 6 minutes
remove and discard the blue Zymo-Midi Filter™ column from the top of the ZymoSpin™ V-E column
transfer the Zymo-Spin™ V-E column to a collection tube and using a microcentrifuge, spin at ≥11,000 x g (or top speed) for 30 seconds to remove any retained lysate
add 400 µl of Endo-Wash Buffer to the Zymo-Spin™ V-E column and centrifuge at ≥11,000 x g (or top speed) for 30 seconds
add 400 µl of Zyppy™ Wash Buffer and centrifuge at ≥11,000 x g (or top speed) for 1 minute. Discard the flow through. Repeat this step and centrifuge at top speed for additional minute to eliminate any residue Wash Buffer from the column
transfer the Zymo-Spin™ V-E column into a clean 1.5 ml microcentrifuge tube and then add 150 µl of Zyppy Elution Buffer to the center of the column. Incubate at room temperature for one minute, then centrifuge at ≥11,000 x g (or top speed) for 1 minute to elute the plasmid DNA
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