DNA Extraction QIAGEN Kit

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For PacBio use the QIAGEN Genomic-Tip 100/G protocol.

  • Grow 10 ml cells to exponential phase final concentration ~10^7 cells/ml in EMM or YES
  • Spin down cells in three steps into one 5ml tube at 2000xg for 2 min.
  • Wash cells from left over media in 5 ml 1X TE (i.e. re-suspend the cells and spin down and remove TE)
  • During the concentration and washing prepare buffer Y1 (in fridge 4)
  • Re-suspend the cells into the isotonic buffer with the MMX lytic enzyme. Make sure all cells are resuspended to optimize the effect of the enzyme.
  • Incubate for 60-120 minutes at 32°C with gentle agitation to generate spheroplasts. (Check at 40X under microscope if spheroplasts can be seen; these are the perfectly round cell sized spheres).
If more than 50% off cells are still intact, increase the incubation time. When working with natural isolates, efficiency can be strain dependent.  
  • When many spheroplast are present, spin down all cells and spheroplasts at >3500xg for 10min at 4°C.

Continue with the protocol from the QIAGEN Blood and Tissue Kit

  • Re-suspend the cells in 180 µl ATL buffer with 20 µl proteinase K (>600 mAU/ml) and incubate for 1h at 56°C
  • Spin down cell debris at >3500xg for 10 min at 4°C.
  • Transfer supernatant to fresh 1.5ml tube
  • Add 200 μl Buffer AL and mix by vortexing.
  • Add 200 μl ethanol (96–100%) mix well by vortexing.
  • Put mixture on a DNeasy Mini column that is in a 2ml collection tube
  • Spin down at 6000 x g for 1 min
  • Place in new collection tube
  • add 500ul AW1
  • Spin down at 6000 x g for 1 min
  • Place in new collection tube
  • add 500ul AW2
  • Spin down at max g for 2 min
  • Discard flow through and spin down again for 1 min
  • Place in a 1.5ml Eppendorf tube.
  • Add 25-50ul AE elution buffer and wait for 5min
  • Spin down at 6000 x g for 1 min
  • Again add 25-50ul AE elution buffer and wait for 5min
  • Spin down at 6000 x g for 1 min

Measure your DNA concentration on the NanoDrop