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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. # Grow yeast cells overnight to exponential phase in 20 ml [[YES]] to a density of ~10⁷ cells/ml. # Spin down cells in a 50ml conical tube at 2000xg for 3 min. # Wash cells in 5 ml 1X [[TE]] (i.e. re-suspend the cells, spin down cells as above and remove TE) #'''work in a fume hood from here on till after elution (step) because buffer Y1 contains the highly toxic beta-mercaptoethanol''' # Prepare in one tube [[Y1 | buffer Y1]] for all samples # 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). # Re-suspend the cells into the Y1+MMX buffer. Make sure all cells are resuspended to optimize the effect of the enzyme. # Incubate for 20 minutes at 30°C with gentle agitation to generate spheroplasts. # Spin down all cells and spheroplasts at >3500xg for 10min at 4°C. # Discard supernatant into a tube or bottle and leave it in the fume hood ('''still toxic'''). # 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. # Incubate for 15 min at 37°C # Add 100 µl protinase K (>600 mAU/ml) and incubate for 1h at 50°C # Spin down cell debris at >3500xg for 10 min at 4°C. # 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 [[buffer QF|QF buffer]] at 50°C # Add the supernatant to the Tip and wait for it to drip through completely. Discard flow through. # Wash by adding 7.5 ml [[QC buffer]] and wait for it to drip through completely # Wash again by adding 7.5 ml [[QC buffer]] and wait for it to drip through completely # '''Transfer the Genomic-Tip to a clean tube''' # Add 5ml of the now warm 50°C elution [[buffer QF]] and wait for it to drip through completely # Add 3.5  ml room temperature pure isopropanol and mix by inverting the tube 10 times to precipitate the DNA. # During this step the DNA should form long threads in the liquid. # Spool the DNA on pipet tip and transfer to 50 µl elution buffer (EB) # 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) # 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. [[Category:Protocols]]

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