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.
- 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 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:
- 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.
- 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.