Team:Ionis Paris/Protocol 9
From the bacterial O/N minicultures, take 500µL that will serve to make bacteria storage at -80°C.
(See the appropriate protocol, Protocol 11 : Glycerol storage) Resuspend the pellet in 200μL Plasmid Resuspension Buffer (B1). Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps. Lyse cells by adding 200μl Plasmid Lysis Buffer (B2). Invert tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for 1 min. Neutralize the lysate by adding 400μl of Plasmid Neutralization Buffer (B3). Gently invert tube until color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 min. Carefully transfer supernatant to the spin column and centrifuge for 1 min. Discard flow-through. Re-insert column in the collection tube and add 200μl of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein and endotoxin. Centrifuge for 1 min. Discard the flow-through. Add 400μl of Plasmid Wash Buffer 2 and centrifuge for 1 min. Transfer column to a clean 1.5 mL microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 min before inserting it into the clean microfuge tube. Add 50μl DNA Elution Buffer to the center of the matrix. Wait for 1 min, then spin for 1 min to elute DNA.
Store the purified DNA at -20°C
Protocol 9: Miniprep
Aim: Extract and purify plasmidic DNA from transformed bacteria
Centrifuge the remaining bacterial O/N mini-cultures (1 to 5 mL) at 13000 rpm for 1 min at room temperature to pellet cells. Discard the supernatant.
Clarify the lysate by spinning for 5 min at 13000 rpm.
