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THE WENDY-PREP - a large-scale modification of the Qiagen Spin Miniprep


The first Qiagen plasmid prep I ever performed was the Maxi Prep, and it sucked. When I got really good plasmid yield from a Qiagen mini-prep spin column, I thought, "why not just do several minipreps instead of a maxi-prep?" The miniprep spin columns take just a fraction of the time it takes to perform a standard Maxi-prep. Furthermore, the cost is about the same (or less): each Maxi-prep costs between $16-$18 (and takes HOURS), while each mini-prep costs just over $1 and only takes about 30 minutes to extract high-quality plasmid DNA.

Using the "Wendy-prep" I've isolated over 500 ug of DNA with just 8 miniprep columns and 50 mls of bacteria. I've also found that you can save a day or so of bacterial growth tme by just inoculating a 50-ml culture directly from a single colony, rather than from a "starter culture."

Large-scale plasmid isolation using the Qiagen Qiaprep Spin Miniprep Kit

Total cost: $15. Total prep time: 30 minutes.

  • Inoculate 50 mls LB + 60 ug/mL ampicillin directly with 1 bacterial colony.
  • Grow 24 hours, shaking, at 30 C.
  • Centrifuge culture 15 minutes at 6000 x g (or 30 minutes at 3000 x g). Remove media.
  • Resuspend pellet in 3 ml P1 buffer. Vortex to disperse pellet completely.
  • Add 3 ml P2 lysis buffer. Mix gently by inverting several times, and lyse 5 minutes at room temperature.
  • Add 4.2 ml N3 neutralizing buffer. Mix thoroughly by inverting, or pipeting and down.
  • Centrifuge at 12,000 x g (or higher) for 10 minutes. If you need to, divide the mixture among several 1.5 ml tubes and centrifuge in a table-top centrifuge.
  • Divide supernatant among 10-12 Qiaprep Miniprep spin columns. Spin ~30 seconds; discard flow-through.
  • Wash each column with 0.7 ml PE wash buffer. Spin; discard flow-through.
  • Spin empty column 1 minute (max speed) to completely remove PE wash buffer.
  • Add 50 ul EB elution buffer (heated to 70 C to increase yield) directly to each column matrix. Let sit for 5 minutes.
  • Spin 1 minute, max speed to elute high quality plasmid.
millions and millions
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