DNA extraction QIAGEN Genomic-Tip S. pombe

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Modified QIAGEN Genomic-Tip 100/G protocol for fission yeast

This method yields high-quality, high-molecular-weight genomic DNA that can be used for cloning and long-read whole genome sequencing with for example MinIon or PacBio.

  1. Grow yeast cells overnight to exponential phase in 20 ml YES to a density of ~107 cells/ml.
  2. Spin down cells in a 50ml conical tube at 2000xg for 3 min.
  3. Wash cells in 5 ml 1X TE (i.e. re-suspend the cells, spin down cells as above and remove TE)
  4. work in a fume hood from here on till after elution (step) because buffer Y1 contains the highly toxic beta-mercaptoethanol
  5. Prepare in one tube buffer Y1 for all samples
  6. In the fume hood add 0.25g Lallzyme MMX powder directly to 4 ml Buffer Y1 and mix until dissolved (this might require some thorough vortexing).
  7. Re-suspend the cells into the Y1+MMX buffer. Make sure all cells are resuspended to optimize the effect of the enzyme.
  8. Incubate for 20 minutes at 30°C with gentle agitation to generate spheroplasts.
  9. Spin down all cells and spheroplasts at >3500xg for 10min at 4°C.
  10. Discard supernatant into a tube or bottle and leave it in the fume hood (still toxic).
  11. Re-suspend the protoplasts in 5 ml buffer G2 (this will lyse them) with 10 µl RNAse (100 mg/ml). Again, re-suspend well to free all the spheroplasts from the pellet.
  12. Incubate for 15 min at 37°C
  13. Add 100 µl protinase K (>600 mAU/ml) and incubate for 1h at 50°C
  14. Spin down cell debris at >3500xg for 10 min at 4°C.
  15. During this centrifugation step:
    • place the genomic tip into its holder and place in clean 50 conical tube
    • prepare the genomic tip columns by adding 4 ml QBT buffer to the Genomic-Tip. This will drip through quickly. Discard flow through
    • place the QF buffer at 50°C
  16. Add the supernatant to the Tip and wait for it to drip through completely. Discard flow through.
  17. Wash by adding 7.5 ml QC buffer and wait for it to drip through completely
  18. Wash again by adding 7.5 ml QC buffer and wait for it to drip through completely
  19. Transfer the Genomic-Tip to a clean tube
  20. Add 5ml of the now warm 50°C elution buffer QF and wait for it to drip through completely
  21. Add 3.5  ml room temperature pure isopropanol and mix by inverting the tube 10 times to precipitate the DNA.
  22. During this step the DNA should form long threads in the liquid.
  23. Spool the DNA on pipet tip and transfer to 50 µl elution buffer (EB)
  24. If spooling is not possible:
    • spin down the DNA
    • Remove isopropanol
    • Wash with -20°C 70% ethanol
    • Air dry
    • Add 50 µl elution buffer (EB)
  25. Re-dissolve the DNA gently into the TE over multiple days at 4°C.

Waste disposal

  • The media from steps 1-4 all need to be collected in the large waste flask in the fume hood (samples are transgenic).
  • Y2 and G2 need to be kept separately because those contain the beta-mercaptoethanol.
  • The flow through QBT, QF and QC are mostly isopropanol and need to be disposed accordingly.