Griffitts:Low-copy plasmid prep

Miniprep procedure for large, low-copy plasmids

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 T₃E_0.3 (preheated to 65°C) directly to disk
  • Don't touch the membrane with your tip
  • Note that T₃E_0.3 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