Team:CSU Fort Collins/Notebook/Protocols=Isolation

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Revision as of 16:48, 17 October 2014

Yeast DNA Isolation

Yeast DNA Isolation Protocol

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  1. Make a 1 cm x 1 cm path of yeast cells in YPD (or appropriate selective media) plates, grow for 1 - 2 days
  2. Cut a microcentrifuge tube at the 100 μL line, but leave a small piece uncut so that you can bend the bottom 90 degrees to make a scoop. Cut a second tube completely and make a funnel. Use the scoop and funnel to add about 100 μL of glass beads to one empty centrifuge tube for each DNA prep
  3. Add 200 μL of DNA prep buffer to each tube as described in TABLE 5-1
  4. TABLE 5-1 HERE
  5. Use a new toothpick to scrape the yeast cells from plates (1 cm x 1 cm patch) and transfer the cells to tubes making a cell/beads/buffer suspension
  6. Add 200 μL of saturated Phenol:Chloroform 1:1 mixture.Vortex at maximum speed for 3 minutes
  7. Let settle for 1 minute, open the tubes carefully and add 200 μL of water or TE
  8. Close again and centrifuge at top speed for 5 minutes
  9. While centrifuging add 10 μL of 4 M ammonium acetate and 1 ml of 100% ethanol to a clean tube
  10. Transfer 300 μL of supernatant from the tubes with ethanol, leave some supernatant behind to avoid collectin the precipitate in the mid layer
  11. Vortex samples briefly to mix. Centrifuge at maximum speed for 5 minutes
  12. Wash the DNA/RNA pellet in 70% ethanol, filling the tube halfway
  13. Discard the 70% ethanol, centrifuge briefly to consolidate the 70% ethanol droplets
  14. Remove the remaining 70% ethanol with a pipette tip or vacuum source
  15. Ressuspend the DNA/RNA pellet in 100 μL of 1X TE
  16. Use 1 - 2 μL of this prep as template for PCR. No RNAse treatment is require for most PCR application

Thank you to Guy Stewart for this protocol.

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