Recovery of Plasmid DNA Using the Wizard© Miniprep Kit
- Grow bacteria in 1-3 ml L-broth or Terrific Broth overnight on a roller at 37°C with appropriate selection. Cell density of the culture is better when Terrific Broth is used.
- Alternatively, a "fake overnight" culture can be prepared by inoculating 1-3 ml of Terrific broth with bacteria from a fresh plate, and allowing the cultures to incubate on a roller at 37°C for 5-7 hours.
- Aliquot one-half of the culture to each of two 1.7-ml microcentrifuge tubes.
- Centrifuge the tubes at high speed in the microcentrifuge for 5 minutes at room temperature to pellet the cells.
- Discard the supernatant, and remove residual liquid with a micropipette.
- Add 200 µl of the cell resuspension solution to one of the two tubes. Resuspend the pellet using the pipette tip, and by pipetting up and down. Recombine the culture (split in step two) by transferring the suspension to the other tube, and resuspend the pellet in that tube in a similar manner.
- Add 200 µl of the cell lysis solution. Mix by inverting gently several times. The suspension should clear almost immediately. If not, continue gentle mixing by inversion until it does.
- Add 200 µl of the neutralization solution. Mix by inverting several times. A white precipitate will form.
- Centrifuge tubes at high speed for 5 minutes.
- Remove the pelleted cell debris using a sterile toothpick, or by decanting the supernatant into a new tube.
- Add 1 ml of well-shaken purification resin to the supernatant saved from step 9. Mix by inverting several times.
- Transfer the slurry to a 3-ml luer-lock syringe to which is attached one of the Wizard© Miniprep columns.
- Transfer the purification resin to the column by slowly depressing the plunger (1 drop/second).
- Detach the syringe from the column, and remove the plunger from the syringe. Reattach the column to the syringe. Pipette 2 ml of the column wash solution (to which has been added 70 ml of 95% ethanol upon opening) into the syringe barrel. Insert the plunger and wash the column by slowly depressing the plunger.
- Transfer the column to a 1.7-ml microcentrifuge tube (it is convenient to use the tube from which the slurry was removed, as this tube will be discarded after this step). Dry the column by centrifuging on high for 20 seconds to 5 minutes (Promega has indicated that a longer spin here results in DNA more suitable for sequence analysis).
- Transfer the column to a new 1.7-ml microcentrifuge tube. Apply 25-50 uµl of dH2O or TE buffer to the column. Allow to stand for one minute. Elute the DNA by centrifuging on high for 20 seconds.
- The amount of dH2O or TE with which to elute depends primarily on plasmid and strain extraction characteristics (e.g. plasmid copy number and size, the ease with which the host strain undergoes lysis, etc.). The use of preheated (65°C) dH2O or TE buffer can sometimes increase recovery while allowing the use of minimal volume in the elution. Repeating the elution step can sometimes improve recovery.
- Store the DNA in the tube at -20°C
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Revised: Sunday, September 03, 1995 1:24:52 PM