Team:Macquarie Australia/plasmid

pbanner

Plasmid Preparation



  1. Centrifuge at 13,200 rpm for 10 min of 1.8mL of overnight cultures in 2mL Eppendorfs.
  2. Discard supernatant and add 1.9 mL of culture and centrifuge again at 13,200 rpm for 10 min.
  3. Re-suspend pelleted cells in P1 Buffer (250µl).
  4. Add 250µL of P2 buffer & invert 4-6 times (turns homogenous blue).
  5. Add 350µL of N3 & mix by inverting (turns colourless).
  6. Centrifuge at 10min 13,000 rpm to obtain a pellet.
  7. Transfer supernatant in QIA Prep Spin Column by pipetting.
  8. Centrifuge 30-60 sec - discard flow through.
  9. Wash QIA Prep Spin Column with 0.5mL of PB.
  10. Centrifuge for 30-60 seconds - discard flow through.
  11. Wash spin column by adding 0.75 mL PE buffer.
  12. Centrifuge for 30-60 seconds - discard flow through and centrifuge for a further 1 min.
  13. Place QIA Prep Column in clean Eppendorf.
  14. Elute DNA by adding 30µl of water, stand for 1 min, centrifuge for 1 min.
  15. Plasmid concentration is measured using NanoDrop 2000 spectrophotometer.