Griffitts:Low-copy plasmid prep

From OpenWetWare

Jump to: navigation, search

Miniprep procedure for large, low-copy plasmids

Contents

Procedure

Lysis

  • Set up an overnight culture in 40 mL LB broth
  • Centrifuge for 8 min at 8000 rpm
  • Resuspend in 2.5 mL of resuspension buffer
    • Alternatively, you can use 2.5 mL old Buffer P1*
  • Add 2.5 mL of lysis solution
    • Alternatively, you can use 2.5 mL old Buffer P2*

DNA Isolation

  • Add 2.5 mL of neutralization solution
    • Alternatively, you can use 2.5 mL old Buffer N3*
  • Centrifuge at 13,000 rpm for 8 min
  • Transfer 6.5 mL of supernatant to 15-mL Falcon tube
  • Add 50 μL of RNAse A
    • If you're using old buffers, you can just leave it on the bench top for 5–10 minutes to allow the native RNAses to work
  • Let sit at RT for 5 min
  • Add 6.5 mL of isopropanol
  • Incubate at –20°C for 30 min
  • Centrifuge in clinical (yellow) centrifuge for 10 min at max speed
  • Pour off supernatant
  • Resuspend pellet in 1 mL 70% ethanol
  • Centrifuge for 5 min in the clinical centrifuge at max speed
    • The clinical centrifuge is the yellow centrifuge by the lab autoclave
  • Discard supernatant

Qiagen Miniprep

  • Resuspend DNA pellet in 250 μL of Buffer P1
    • The P1 is kept in the fridge
    • Make sure to use P1 that has RNAse added
  • Add 250 μL of Buffer P2
  • Immediately invert 3 times
  • Let stand for 2 minutes
  • Add 350 μL of Buffer N3
  • Immediately invert 8 times
  • Keep on ice for 5 min
  • Centrifuge at 13,000 rpm for 6 min in microfuge
  • 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 70 μL T3E0.3 (preheated to 65°C) directly to disk
    • Don't touch the membrane with your tip
    • Note that T3E0.3 must be added to the column hot for efficient elution of large (20 kb) plasmids
  • Let stand 1–2 minutes
  • Centrifuge for 60 seconds at full speed to elute DNA

*The old buffers are kept in a Qiagen box on the upper shelf

Personal tools