Griffitts:Low-copy plasmid prep

Miniprep procedure for large, low-copy plasmids

Procedure

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^*

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

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 Griffitts:Common buffers#TE buffer (preheated to 65°C) directly to disk
- Don't touch the membrane with your tip
- Note that Griffitts:Common buffers#TE buffer 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