Griffitts:Miniprep

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Standard Qiagen Miniprep

Materials

  • Qiaprep Spin Kit (27106)
  • TE buffer
  • 1.5 mL microcentrifuge tubes (1 per culture)

Procedure

  • Grow DH5α with appropriate plasmid in 4 mL liquid LB+antibiotic for 12–18 hours
  • Label your spin columns and microcentrifuge tubes
  • Centrifuge 1.6 mL of each culture in a microcentrifuge tube at full speed for 30 seconds
  • Dump and tap out supernatant
  • Resuspend pellet in 250 μL P1
    • The P1 is kept in the fridge
    • Make sure to use P1 that has RNAse added
  • Add 250 μL P2
  • Immediately invert 3 times
  • Let stand for 2 minutes
  • Add 350 μL N3
  • Immediately invert 8 times
  • Centrifuge for 6 minutes at full speed
  • Carefully move the supernatant (~820 μL) to a spin column
  • Centrifuge for 30 seconds at full speed
  • Remove flowthrough
  • Add 700 μL of wash buffer PE to column
  • Centrifuge for 30 seconds at full speed
  • Remove flowthrough
  • Centrifuge for another 60 seconds at full speed
  • Move column to a labeled 1.5 mL microcentrifuge tube
  • Add 50–100 μL TE directly to disk
    • Don't touch the membrane with your tip
  • Let stand 1–2 minutes
  • Centrifuge for 60 seconds at full speed to elute DNA

Adapted from Qiagen handbook

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