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1. Add the provided RNase A solution to Buffer P1, mix, and store at 4C. (One vial of RNase A per bottle of Buffer P1 to give final concentration of 100ug/mL. If you're the one adding, initial top and check box on cap. Buffer P1 will be in the fridge)
2. Optional: Add LyseBlue reagent to Buffer P2 at a ratio of 1 to 1000 and mix (If you're the one adding, initial top and check the box on cap)
3. Add ethanol (96-100%) to Buffer PE before use (see bottle label for volume) and then check mark on cap.
4. Check Buffers P2 and N3 for precipitates, if any redissolve by placing in water bath at 37C. Do NOT vortex.

STEPS:

1. Harvest bacterial culture by centrifuging at 6000 x g for 15 min at 4C (in 50ml conicals).
2. Completely resuspend pelleted bacteria in 4 ml 4ml Buffer P1.
3. Add 4 ml 4ml Buffer P2, gently mix by inverting until the lysate appears viscous, and incubate at room temperature (15-25C) for 3 min. If LyseBlue reagent had been added, the cell suspension will turn blue.
4. Place the QIAfilter Cartridge into a new and suitable tube, allowing space for addition of Buffer BB.
5. Add 4 ml Buffer S3 to the lysate, and mix by inverting 4-6 times. If LyseBlue reagent has been added, mix the solution until it is completely colorless.
6. Transfer the lysate to the QIAfilter Cartridge and incubate at room temperature for 10 min. The DNA is now stable; this is an appropriate place in the protocol to pause (if necessary).
7. During incubation, place QIAGEN plasmid Plus spin columns into the QIAvac 24 Plus. Insert Tube Extenders into each column.
8. Gently insert the plunger into the QIAfilter Cartridge and filter the cell lysate into the tube.
9. Add 2 ml Buffer BB to the cleared lysate, and mix by inverting 4-6 times.
10. Transfer lysate to a QIAGEN Plasmid Plus spin column on the QIAvac 24 plus.
11. Apply approximately -300 mbar vacuum until the liquid has been drawn through all columns.
12. To wash DNA, add 0.7 ml Buffer ETR and apply vacuum until the liquid has been drawn through all columns.
13. To further wash the DNA, add 0.7 ml Buffer PE and apply vacuum until the liquid has been drawn through all columns.
14. To completely remove the residual wash buffer, centrifuge the column at 10,000 x g (9,700 rpm) for 1 min in a tabletop microcentrifuge.
15. Place the QIAGEN Plasmid Plus spin column into a clean 1.5 ml tube. To elute the DNA, add 200 ul Buffer EB or water to the center of the QIAGEN Plasmid Plus spin column, let it stand for > 1 min, and centrifuge for 1 min.