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Genomic DNA Purification - Yeast

  1. Grow yeast culture (1.5 -3.0 ml) in YPD medium to an appropriate OD600.

  2. Harvest no more than 3 ml culture (< 2 x 107) by centrifugation at 4,000 x g for 10 min at room temperature. 

  3. Discard medium and resuspend cells in 480 µl Buffer SE and 20 µl lyticase solution. Incubate at 30oC for at least 30 min.

  4. Pellet spheroblasts by centrifuging at 500 x g for 10 min at room temperature.

  5. Resuspend cells in 200 µl Buffer YL. Add 50 mg glass beads and vortex for 5 min. Buffer YL and glass beads can be mixed before experiment. Using glass beads gives best quality and yield of gDNA.

  6. Add 20 µl Proteinase K solution (5 mg/ml) and vortex to mix well. Incubate at 55oC in a shaking water bath till complete lysis. Lysis times varies depending on the species and amount of yeast and is usually complete in 15 - 60 min. Overnight lysis is OK. If no shaking water-bath is available, incubate and shake or briefly vortex the samples every 20-30 min.

  7. Add 5 µl RNase A (10 mg/ml) to samples and invert tube several times to mix. Incubate at room temperature for 5 min. (Optional) Centrifuge at 10,000 x g for 5 min to pellet insoluble debris. Carefully aspirate the supernatant and transfer to a sterile micro-centrifuge tube leaving behind any insoluble pellet.

  8. Add 220 µl Buffer YBL and vortex to mix at maximum speed for 15 s. Incubate at 65oC for 10 min. A wispy precipitate may form upon addition of Buffer YDL; it does not interfere with DNA recovery.

  9. Add 220 µl absolute ethanol (room temperature, 96-100%) and mix thoroughly by vortexing at maxi speed for 20 s. If any precipitation can be seen at this point, break the precipitation by pipetting up and down 10 times.

  10. Transfer the entire sample into the column, including any precipitate that may have formed. Centrifuge at 10,000 x g for 1 min. Discard the collection tube and filtrate.

  11. Place the column in a new 2 ml tube.  Add 500 µl Buffer KB. Centrifuge at 10,000 x g for 1 min. Discard flow-through and reuse the collection tube.

  12. Add 700 µl DNA Wash Buffer to the column. Centrifuge at 10,000 x g for 1 min. Discard flow-through and reuse the collection tube. Ensure that ethanol is added to the DNA wash buffer before use.

  13. (Optional): Repeat Step 12.

  14. Centrifuge the column at maximum speed (10,000 x g) with the lid open for 2 min. This step is critical for removal of trace ethanol that might otherwise interfere with down stream applications.

  15. Place the column in a nuclease-free 1.5 ml microtube. Add 50-100 µl TE Buffer or water to column. Incubate for 1 min. Centrifuge at 10,000 x g for 1 min to elute DNA. Using preheated (65oC) TE Buffer or water can increase DNA yield. Incubating the column at 65oC rather than at room temperature prior to centrifugation will give a modest increase in DNA yield per elution.

  16. Repeat the elution with a second 50-100 µl TE Buffer or water if more DNA is needed.

     

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