Griffitts:Miniprep

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 liquid LB (4 mL) for 12-18 hours
  • Add the appropriate antibiotics if necessary
• Label your spin columns and microcentrifuge tubes
• Centrifuge 1.3 mL of culture at full speed for 30 seconds
• Dump and tap out supernatant
• Add another 1.3 mL of culture
• Spin at full speed for 30 seconds
• 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 650 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