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 ) .
