• Created by Unknown User (shinjini@mit.edu), last modified by Unknown User (lylaatta@mit.edu) on May 06, 2015 12:54

Before you Start:

  1. Optional: Add LyseBlue reagent to Buffer P1 at a ratio of 1 to 1000 and mix (If you're the one adding, initial top and check the box on cap)
  2. Add the provided RNase A solution to Buffer P1, mix, and store at 4C. (One vial of RNase A per bottle of Buffer P1 to give final concentration of 100ug/mL. If you're the one adding, initial top and check box on cap. Buffer P1 will be in the fridge)
  3. Add ethanol (96-100%) to Buffer PE before use (see bottle label for volume) and then check mark on cap.
  4. Check Buffers P2 and N3 for precipitates, if any redissolve by placing in water bath at 37C Do NOT vortex.

Steps

  1. Pellet 1-5mL of bacterial overnight culture by centrifugation at >8000 rpm for 3 min at room temperature (15-25C; use 2ml microcentrifuge collection tubes). Decant all the liquid and add 1 ml of the culture into the corresponding tube. Make sure not to mix up the tries.
  2. Resuspend pelleted bacterial cells in 250 uL Buffer P1 and transfer to microcentrifuge tube.
  3. Add 250uL Buffer P2 and mix thoroughly by inverting tube 4-6 times. Do NOT vortex. Mixture turns blue. Do NOT allow this lysis reaction to proceed for more than 5 min.
  4. Add 350uL of Buffer N3 and mix IMMEDIATELY and thoroughly by inverting tube 4-6 times. Do NOT vortex. Mixture is now colorless.
  5. Centrifuge for 10min at 13,000 rpm in table-top centrifuge.
  6. Apply the supernatant to a QIAprep spin column by decanting or pipetting. Do NOT get any of the sticky precipitate.
  7. Centrifuge for 30 - 60s at 13000rpm. Discard flow-through.
  8. Wash the QIAprep column by adding 0.5 mL Buffer PB.
  9. Centrifuge for 30 - 60s at 13000rpm. Discard flow-through.
  10. Wash the QIAprep column by adding 0.75 mL Buffer PE.
  11. Centrifuge for 30 - 60s at 13000rpm. Discard flow-through.
  12. Centrifuge for 1 min to remove residual was buffer.
  13. Place the QIAprep column in a clean 1.5mL microcentrifuge tube. To elute DNA, add 50uL Buffer EB to center of each column. Be careful NOT to pierce column.
  14. Let stand for 1 minute.
  15. Centrifuge for 60s at 13000rpm.
  16. Remove column and discard, tube now contains DNA.
  17. Go to NanoDrop and spec DNA.

 

http://2014.igem.org/wiki/images/f/f5/QIAprep-Spin-Miniprep-Kit-EN.pdf

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