Qiagen’s QIAprep Miniprep Kit is based on alkaline lysis of bacteria followed by adsorption onto a silica-gel-membrane in the presence of high salt. This process streamlines and eliminates many steps, such as phenol extraction or ethanol precipitation, from more traditional mini-prep protocols. The kit provides a reliable and fast preparation for small quantities of plasmid DNA (up to 20 ug). The only equipment required is a tabletop centrifuge to pellet bacteria and a microcentrifuge for clearing lysates and elution of DNA. The QIAprep Spin Miniprep Kit is suitable for processing 1-24 samples in less than 30 min., however, variations on the system are available which allow for a higher throughput. For example, the Qiaprep 8 Miniprep Kit allows the user to process 48 samples in 40 min and the QIAprep 96 Turbo Miniprep Kit enables the processing of up to 96 samples in 45 min.
The first step of the protocol is overnight culture of bacteria in 1-5 ml of LB medium. The bacteria are then harvested by centrifugation and the supernatant is removed. The pellet is re-suspended in 250 ul of buffer P1 (Tris-EDTA buffer) and transferred to a microcentrifuge tube. Once re-suspended, 250 ul of buffer P2 (lysis buffer - 200mM NaOH, 1%SDS) is added to the suspension and the tube is mixed by inversion 6 times. At this point, the tube is incubated for 5 min. at room temperature. Following incubation, 350 ul of buffer N3 (neutralization buffer) is then added, the tube is inverted 6 times and immediately centrifuged for 10 min. at 13000 rpm. After the previous step, the procedure can continue using centrifugation or using the Qiagen vacuum manifold. We have generally used the vacuum manifold and, therefore, we decant the supernatant from the centrifuged tube into the QIAprep spin column (which is inserted onto the luer extensions on the vacuum manifold). Vacuum is applied to the manifold, the supernatant is drawn through the QIAprep spin column and 0.75ml of wash buffer PE is applied to the QIAprep spin column (also drawn through by re-applying the vacuum). The QIAprep spin column is then removed from the vacuum manifold, placed in a collection tube and microcentrifuged for 1 min. to remove residual wash buffer. Plasmid DNA is eluted from the membrane of the QIAprep spin column by adding a small volume of tris buffer or water (50 ul). The QIAprep spin column is placed in a fresh microcentrifuge tube and water or buffer is added. This is incubated on the membrane for 1 min. prior to centrifugation for 1 min. to elute the DNA from the membrane. The DNA is then ready for subsequent applications (i.e. sequencing, site directed mutagenesis, etc.). We generally perform the last step with water, as this allows the purified DNA to be used easily in various applications requiring different type of buffers.
This kit is fast, reliable and produces a good yield of quality plasmid DNA from small volumes of bacteria, meeting the demands of most downstream applications, such as sequencing. For those reasons, this is our plasmid miniprep kit of choice for preparation of plasmid DNA from small bacterial cultures.
Boyd Scott
Senior Research Scientist
Discovery Biology
Vitae Pharmaceuticals