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For PacBio use the [[QIAGEN Genomic-Tip | 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) ** In a fume/safety hood (buffer contains the highly toxic beta-mercaptoethanol) add 50mg [[Lallzyme MMX]] ([http://dx.doi.org/10.1002/yea.2994 Flor-Parra et al 2014, doi:10.1002/yea.2994]) powder directly to 0.5 ml [[Y1 | Buffer Y1]] and mix until dissolved (no sterilization required). * 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 [[DNA concentration with the NanoDrop | NanoDrop]] [[Category:Protocols]]

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