IGEM:UC Berkeley/2006/midipreps

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A midiprep is for production of ~100 ug DNA from 50 (high copy) to 500 mL (low copy) of cells. Qiagen has multiple "midi" kits, the most useful of which is the HiSpeed variant, but the Qiafilter one is good too. The procedure begins with a saturated culture of bacteria in a flask:

  1. transfer cells to a 50mL conicol, pellet at 8000 rpm for 4 min (in Keasling lab Allegra)
  2. Resuspend pellet in 4mL P1 + 8mL millipore water. Vortex to remove all clumps.
  3. Lyse with 12mL of P2, mix gently by inverting several times
  4. Neutralize with 12mL of cold P3 ***not N3, it won't work***
  5. Swirl and invert until it reach egg drop state, then shake to miso
  6. Spin for 10min in the Allegra at 8000 rpm. This won't entirely remove the debris, but gets most of it down
  7. Meanwhile, add 5mL QBT to the column. As with all column steps, let it drip through by gravity, don't force it. Also, let the liquid flow all the way through until the frit looks dry
  8. Using the syringe filter provided with the kit, dump in the lysate, and push it through onto the column. You likely will have to repeat this to get all the liquid through the filter.
  9. Let flow through 10mL of QC ***make sure you are using QC and not QF***
  10. Do another 10mL of QC
  11. Elute column with 5mL QF into a 15 mL conicol tube
  12. Add 3.5mL of isopropanol, shake to mix. This precipitates the DNA, but you probably can't see it yet.
  13. Load the mixture into the round 0.2um filters attached to a syringe. Slowly push through the liquid. At this point, your precipitated DNA is stuck in the filter.
  14. Remove the syringe, load with air, blow it through the filter a few times to get most of the liquid out, but it doesn't have to be perfectly dry--a little isopropranol will aid resuspension.
  15. Elute the syringe with 1mL of TE buffer into a 2mL eppendorf
  16. Add 100uL of 3M sodium acetate pH 5.5 and 800uL of isopropanol to the tube. Invert several times. The DNA pellet should appear at this point a white snot-like wisp in the tube. The sodium acetate solution is made by mixing equal moles of acetic acid and sodium acetate.
  17. microentrifuge full speed for 5 min, dump supernatant
  18. Add 1mL 4degree 70% ethanol, invert gently, try not to disrupt the pellet
  19. Spin 1 min full speed to re-seat the pellet, dump supernatant
  20. Repeat the 70% ethanol wash
  21. Add 1mL of 4 degree 100% ethanol, invert, spin 1min, dump supe. This removes the remaining traces of water from the pellet making it dry faster and also resuspend better in the last step. ***Be very careful, the dried pellet doesn't stick to the tube very well.
  22. Dry in the 37 oven until there is no apparent liquid (~5 min usually)
  23. Resuspend in whatever volume of water you want, I usually adjust this for the size of the pellet, but 100 uL is a good ballpark number for a very concentrated prep.