Qiagen MidiPrep Plus
1. Grow bacteria in 250 ml LB/Amp or LB/kan overnight at 37 oC.
Spin down cells in 500 ml centrifuge tube. Add some deionized water, resuspend, transfer cells to a clean 50 ml VWR centrifuge tube. Repeat washing the 500 ml centrifuge tubes 2 times. Transfer the liquid to the 50 ml centrifuge tube. Spin for 5 min at 3000 rpm. Decant liquid.
2. Resuspend pelleted bacteria in 2 ml or 4 ml Buffer P1.
Ensure that RNase A has been added to Buffer P1 (see âThings to do before startingâ on page 18 for all protocols).
For efficient lysis, it is important to use a vessel that is large enough to allow complete mixing of the lysis buffers. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
3. Add 2 ml or 4 ml Buffer P2, gently mix by inverting, and incubate at room temperature for 3 min.
The lysate should appear viscous. Do not allow lysis to proceed for more than 5 min. After use, the bottle containing Buffer P2 should be closed immediately to avoid acidification from CO2 in the air.
If LyseBlue has been added to Buffer P1, the cell suspension will turn blue after addition of Buffer P2. Mixing should result in a homogeneously colored suspension. If the suspension contains localized colorless regions or if brownish cell clumps are still visible, continue mixing the solution until a homogeneously colored suspension is achieved.
4. Add 2 ml or 4 ml Buffer S3 to the lysate and mix immediately by inverting 4â6 times. Proceed directly to step 4. Do not incubate the lysate on ice.
After addition of Buffer S3, a fluffy white precipitate containing genomic DNA, proteins, cell debris, and SDS becomes visible. It is important to transfer the lysate into the QIAfilter cartridge immediately in order to prevent later disruption of the precipitate layer. If the mixture still appears viscous and brownish, more mixing is required to completely neutralize the solution. If LyseBlue reagent has been used, the suspension should be mixed until all trace of blue has gone and the suspension is colorless. A homogeneous colorless suspension indicates that the SDS has been effectively precipitated.
5. Spin at 3000 rpm at room temperature for 10 min. Transfer the supernatant lysate to the QIAfilter cartridge and incubate at room temperature for 10 min.
If working with low-copy plasmid culture volumes, it may be beneficial to centrifuge the lysate before clarifying through QIAfilter cartridge. The centrifugation is performed instead of the 10 min incubation and leads to convenient filtration without clogging. The centrifugation can be performed directly in the same vessel which is used for alkaline lysis if this vessel is appropriate for centrifugation. Centrifugation should be performed for 5 min at approx. 4500 x g. Minor amounts of the precipitated material will be on the surface of the supernatant and can easily be removed with a pipette tip while pouring the supernatant into the QIAfilter cartridge.
Important: Room-temperature incubation is essential for optimal performance of the QIAfilter cartridge. Do not agitate the QIAfilter cartridge during this time. A precipitate containing proteins, genomic DNA, and detergent will float and form a layer on top of the solution. This ensures convenient filtration without clogging. If, after the incubation, the precipitate has not floated to the top of the solution, carefully run a sterile pipet tip around the walls of the cartridge to dislodge it.
6. During incubation, prepare the vacuum manifold and the QIAGEN Plasmid Plus Midi spin columns.
7. Gently insert the plunger into the QIAfilter cartridge and filter the cell lysate into a new tube, allowing space for the addition of Buffer BB. Filter until all of the lysate has passed through the QIAfilter cartridge, but do not apply extreme force.
8. Add 2 ml Buffer BB to the cleared lysate and mix by inverting 4â6 times. You should see: the solution becomes milky turbid. If you forget to add Buffer BB, you are either stupid or careless, you will be fired.
9. Transfer lysate to a QIAGEN Plasmid Plus Midi spin column with a tube extender attached on the QIAvac 24 Plus.
10. Switch on the vacuum source, apply approx. â300 mbar, and draw the solution through the QIAGEN Plasmid Plus Midi spin column.
After the liquid has been drawn through all columns, switch off the vacuum source. Closing VacValves after liquid has drawn through the first columns ensures that the vacuum pressure remains constant and stable for the remaining samples.
11. To wash the DNA, add 0.7 ml Buffer ETR and proceed with one of the following two steps:
Washing using a vacuum:
After adding 0.7 ml Buffer ETR, switch on the vacuum source. After the liquid has been drawn through all columns, switch off the vacuum source.
Washing using a microcentrifuge:
Discard the tube extenders and place the QIAGEN Plasmid Plus Midi spin column into the 2 ml collection tube provided. Wash the column by adding 0.7 ml Buffer ETR and centrifuging for 1 min at 10,000 x g. Discard the flow-through.
12. To further wash the DNA, add 2 X 0.35 ml Buffer PE and proceed with one of the following two steps:
Washing using a vacuum:
After adding 0.7 ml Buffer PE, switch on the vacuum source. After the liquid has been drawn through all columns, switch off the vacuum source.
Washing using a microcentrifuge:
Place the QIAGEN Plasmid Plus Midi spin column in the empty collection tube from step 12. Wash the DNA by adding 2X 0.35 ml Buffer PE and centrifuging for 1 min at 10,000 x g. Discard the flow-through.
13. To completely remove the residual wash buffer, centrifuge the column for 1 min at 10,000 x g (13000 rpm) in a microcentrifuge.
14. Place the QIAGEN Plasmid Plus Midi spin column into a clean 1.5 ml microcentrifuge tube. To elute the DNA, add 200 ÎŒl of Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the QIAGEN Plasmid Plus Midi spin column, let it stand for at least 1 min, and centrifuge for 1 min. Water or buffers commonly used to dissolve DNA (e.g., TE) may also be used for elution.