NOTEBOOK

Extraction protocol of plasmid DNA (Miniprep)

For extraction of plasmid DNA was used Qiagen Spin Miniprep Kits.

Materials: Qiagen Miniprep Kit; Cell culture; 1.5 ml microtubes.

• 1) Bacterial culture Centrifuge at 3000 g for 10 minutes.
• 2) Discard supernatant containing sanitary water container.
• 3) Resuspend pellet of bacterial cells in 250 ul of buffer P1. Transfer to a 1.5 ml tube.
• 4) Add 250 uL of Buffer P2 and inverting the tube gently 4-6 times to mix. Not exceed five minutes with the lysis reaction.
• 5) Add 350 ul of buffer N3 and invert the tube immediately and gently 4-6 times.
• 6) Centrifuge for 10 minutes at 13,000 rpm in a microcentrifuge.
• 7) Apply the supernatant from step 4 on QIAprep spin column by pipetting.
• 8) Centrifuge for 30-60 s.
• 9) Wash QIAprep spin column by adding 0.75 ul of buffer PE and centrifuged for 30-60 s. Remascente discard the liquid.
• 10) Centrifuge for an additional 1 min to remove residual washing buffer.
• 11) Place the QIAprep column in a clean 1.5 ml microfuge tube.
• 12) For eluting the DNA, add 50 ul Buffer EB (10 mM Tris-HCl, pH 8.5) or water in the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.