Purification of Plasmid DNA by Miniprep

Centrifuge 1-5mL of the overnight LB-culture. Remove all medium.

Add 250µL Resuspension Buffer (R3) with RNase A to the cell pellet and resuspend the pellet until it is homogeneous

Add 250µL Lysis Buffer (L7). Mix gently by inverting the capped tube until the mixture is homogeneous. Do not vortex. Incubate the tube at room temperature for 0h 5m 0s.

Add 350µL Precipitation Buffer (N4). Mix immediately by inverting the tube, or for large pellets, vigorously shaking the tube, until the mixture is homogeneous. Do not vortex. Centrifuge the lysate at >12,000 × g for 0h 10m 0s

Load the supernatant from step 4 onto a spin column in a 2-mL wash tube. Centrifuge the column at 12,000 × g for0h 1m 0s. Discard the flowthrough and place the column back into the wash tube.

Add 500µL Wash Buffer (W10) with ethanol to the column. Incubate the column for 0h 1m 0s at room temperature. Centrifuge the column at 12,000 × g for 0h 1m 0s. Discard the flowthrough and place column back into the wash tube

Add 700µL Wash Buffer (W9) with ethanol to the column. Centrifuge the column at 12,000 × g for 0h 1m 0s. Discard the flowthrough and place the column into the wash tube. Centrifuge the column at 12,000 × g for 0h 1m 0s. Discard the wash tube with the flowthrough

Place the Spin Column in a clean 1.5-mL elution tube. Add 75µL of preheated TE Buffer (TE) to the center of the column. Incubate the column for 0h 1m 0sat room temperature.

Centrifuge the column at 12,000 × g for 2 minutes. The elution tube contains the purified plasmid DNA. Discard the column. Store plasmid DNA at 4°C (short term) or store the DNA in aliquots at −20°C (long term).