Griffitts:Low-copy plasmid prep

Miniprep procedure for large, low-copy plasmids

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