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Team:CSU Fort Collins/Notebook/Protocols=Isolation
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Revision as of 16:48, 17 October 2014
Yeast DNA Isolation Protocol
Show Table of Contents
- Make a 1 cm x 1 cm path of yeast cells in YPD (or appropriate selective media) plates, grow for 1 - 2 days
- 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
- Add 200 μL of DNA prep buffer to each tube as described in TABLE 5-1 TABLE 5-1 HERE
- 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
- Add 200 μL of saturated Phenol:Chloroform 1:1 mixture.Vortex at maximum speed for 3 minutes
- Let settle for 1 minute, open the tubes carefully and add 200 μL of water or TE
- Close again and centrifuge at top speed for 5 minutes
- While centrifuging add 10 μL of 4 M ammonium acetate and 1 ml of 100% ethanol to a clean tube
- Transfer 300 μL of supernatant from the tubes with ethanol, leave some supernatant behind to avoid collectin the precipitate in the mid layer
- Vortex samples briefly to mix. Centrifuge at maximum speed for 5 minutes
- Wash the DNA/RNA pellet in 70% ethanol, filling the tube halfway
- Discard the 70% ethanol, centrifuge briefly to consolidate the 70% ethanol droplets
- Remove the remaining 70% ethanol with a pipette tip or vacuum source
- Ressuspend the DNA/RNA pellet in 100 μL of 1X TE
- Use 1 - 2 μL of this prep as template for PCR. No RNAse treatment is require for most PCR application
