Promega Wizard® Plus Miniprep Protocol


The following steps describe a standard plasmid miniprep using the Magic Minipreps(TM) system starting from a 1-3ml culture of E. coli.

A. Production of Cleared Lysate

  1. Pellet 1-3ml of cells by centrifugation (13,000g for 1 min). Resuspend the cell pellet in 200ul of Cell Resuspension Solution. Vortex.
  2. Add 200ul of FRESH Cell Lysis Solution (see below) and mix by inverting the tube several times. The cell suspension will clear almost immediately. If it does not, continue inverting until it clears.
  3. Add 200ul of Neutralization Solution and mix by inverting the tube several times.
  4. Spin in a microcentrifuge at 13,000g for 6 minutes.
  5. Decant the cleared supernatant to a new microcentrifuge tube.

 

B. Plasmid Purification Using a Disposable Syringe (Without a vacuum)

  1. Add 1ml of Magic Minipreps DNA Purification Resin to the supernatant from step A.5 and mix by inverting the tube.
  2. For each miniprep, use one Magic Minipreps mini-column. Attach a 3ml disposable syringe barrel to the luer-lok extension of each mini-column.
  3. Pipet the Magic Minipreps DNA Purification Resin containing the bound DNA into the syringe barrel. Insert the plunger and gently push the slurry into the mini-column with the syringe plunger.
  4. Wash the mini-column with 2ml Column Wash Solution by removing the mini-column from the syringe and taking up the solution in the syringe. Reattach the syringe to the mini-column and gently push the Column Wash Solution through the mini-column with the syringe plunger.

    *** Important: The column wash solution is not supplied with ethanol. Prior to use, sufficient absolute ethanol must be added to the column wash solution to bring the final concentration to 50%(v/v).
  5. Transfer the mini-column to a microcentrifuge tube, and place in a microcentrifuge. Spin the mini-column for 20 seconds at 10,000g to dry the resin.
  6. Transfer the mini-column to a new microcentrifuge tube.
  7. To elute the plasmid DNA, apply to the mini-column 50ul of water or TE buffer preheated to 65-70°C.
  8. Spin the microcentrifuge tube containing the mini-column for 30 seconds at 10,000g in a microcentrifuge. Remove and discard the mini-column. Plasmid DNA may be stored in the microcentrifuge tube at 4° or -20°C. Each Magic Minipreps DNA isolation will yield up to 10ug of plasmid DNA.

Cell Lysis Solution
0.2N NaOH 0.4ml 5N NaOH
1% SDS 1ml 10% SDS
ddH20 to 10ml 8.6ml ddH20
TOTAL 10ml