DNA from cultured cells

From QIAamp DNA Mini & Blood Mini Handbook, Apendix B, p 50

Do not use more than 5 x 10^6 cells

Important points before starting

• Do not use more than 5 x 106 cells (with a normal set of chromosomes).
• All centrifugation steps are carried out at room temperature (15–25°C).
• Use carrier DNA if the sample contains <10,000 genome equivalents (see page 17).

Things to do before starting

• Heat a water bath or heating block to 56°C.
• Equilibrate Buffer AE or distilled water to room temperature (15–25°C) for elution.
• Ensure that Buffer AW1, Buffer AW2, and QIAGEN Protease have been prepared according to the instructions on page 16.
• If a precipitate has formed in Buffer AL, dissolve by incubating at 56°C.

Procedure

• Harvest cells
  • Trypsinize and count cells
  • Put no more than 5 million cells into a microfuge tube
  • Centrifuge for 5 min at 300 x g.
  • Remove the supernatant completely and discard, taking care not to disturb the cell pellet.
• Resuspend cell pellet in PBS to a final volume of 200μL.
• Add 20 μL QIAGEN Protease or proteinase K.
• (Continue with step 3 of “Protocol: DNA Purification from Blood or Body Fluids (Spin Protocol)”, page 27)