Modified Qiagen Plasmid Midi
Harvest overnight bacterial culture by centrifuging at 4000 x g for 15 min at 4°C.
Resuspend the bacterial pellet in 4 ml Buffer P1.
Add 4 ml Buffer P2, mix thoroughly by vigorously inverting 4–6 times, and incubate at room temperature (15–25°C) for 5 min.
If using LyseBlue reagent, the solution will turn blue.
Add 4 ml prechilled Buffer P3, mix thoroughly by vigorously inverting 4–6 times.
Incubate on ice for 15 min.
If using LyseBlue reagent, mix the solution until it is colorless.
Centrifuge at 14,000 x g for 45 min at 4°C.
Equilibrate a QIAGEN-tip 100 by applying 4 ml Buffer QBT, and allow column to empty by gravity flow.
Apply the supernatant from step 5 to the QIAGEN-tip and allow it to enter the resin by gravity flow.
Wash the QIAGEN-tip with 2 x 10 ml Buffer QC.
Allow Buffer QC to move through the QIAGEN-tip by gravity flow.
Elute DNA with 5 ml Buffer QF into a clean 15 ml vessel.
For constructs larger than 45 kb, prewarming the elution buffer to 65°C may help to increase the yield.
Precipitate DNA by adding 3.5 ml room-temperature isopropanol to the eluted DNA and mix.
Centrifuge at 4,000 x g for 60 min at 4°C.
Carefully decant the supernatant.
Wash the DNA pellet with 2 ml room-temperature 70% ethanol and centrifuge at 4,000 x g for 10 min.
Carefully decant supernatant.
Air-dry pellet for 5–10 min and redissolve DNA in a suitable volume of appropriate buffer (e.g., TE buffer, pH 8.0, or 10 mM Tris·Cl, pH 8.5).
