Paul.xu at 23:05, 28 June 2014

2014-06-28T23:05:34Z

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== Quick Yeast Genomic DNA Prep ==

 1) Grow 10 ml yeast cultures to saturation in YEPD. 2) Pour the culture into a 15 ml conical and spin at 3000 rpm for 3 min. Dump sup. Resuspend in 500 µl distilled water. 3) Transfer to microfuge tube. Spin at 13000 rpm for 1 min. Dump sup. Vortex briefly to resuspend the pellet in the residual liquid. 4) Add 200 µl detergent lysis buffer, 200 µl phenol chloroform (at 4°C, take bottom layer), and ~300 mg glass beads (1 scoop). 5) Vortex 3-4 minutes on multihead vortex. 6) Add 200 µl TE pH 8. Spin at 13000 rpm for 5 minutes. Transfer aqueous layer (top) to a fresh tube. 7) Add 1 ml of ice cold 100% EtOH. Mix by inversion. 8) Spin at 13000 rpm for 2 minutes at 4°C. Dump sup. Resuspend in 400 µl TE pH 8 and 3 µl RNase. 9) Incubate 5 minutes at 37°C. 10) Add 10 µl 4 M ammonium acetate plus 1 ml of ice cold 100% EtOH. Mix by inversion 11) Spin at 13000 rpm for 2 minutes at 4°C. Dump sup. Air-dry pellet. 12) Resuspend in 50 µl TE. 13) Store at -20°C.

=== Materials: ===

 -Saturated culture -Detergent lysis buffer: 2% Triton X 100 1% SDS 100 mM NaCl 10 mM Tris-Cl pH 8 1 mM EDTA -PCI: Phenol, chloroform, IAA (25:24:1) -acid-washed glass beads -TE pH 8 -10 mg/ml boiled RNase -4 M ammonium acetate 

== Genomic DNA Extraction S. cerevisiae and S. pombe ==

=== Protocol: ===

 1. Grow a 10ml overnight culture, spot onto YPD to check markers 2. Pellet cells, decant supernatant and resuspend in 0.5ml water 3. Transfer to a 1.7ml micro-centrifuge tube 4. Pellet 5 seconds and decant supernatant 5. Briefly vortex pellet to resuspend in remaining medium 6. Add: 0.2ml Smash and Grab Solution 0.2ml Phenol-Chloroform 0.3grams Acid-washed glass beads 7. Vortex for 2-5 minutes using the multi-tube holder or 10 minutes on multi-tube vortexer in the cold room 8. Add 0.2ml of TE to each tube 9. Spin 5 minutes in centrifuge 10. Transfer aqeous phase to new tube 11. Add 1.0ml EtOH and mix to ethanol precipitate 12. Spin down 5 minutes, decant supernatant 13. Resuspend pellet in 0.4ml TE 14. Add 30μg RNase and incubate for 5 minutes at 370 15. Add 10μl 4M ammonium acetate and 1.0ml EtOH 16. Ethanol precipitate on ice for 5 minutes 17. Spin down 10 minutes 18. Optional wash with 0.5ml 70% EtOH 19. Air dry pellet and resuspend in 100μl TE Entered by Diana Libuda on 12/6/2007

=== Smash and Grab Solution (100mL) ===

 10 mL 10% SDS (1%) 2 mL 100x Triton-X100 (2%) 2 mL 5M NaCL (100mM) 1 mL 1M Tris pH 8.0 (10mM) (1mM) Mix in beaker, adding the detergents while the solution is mixing. Filter sterilize.

[[Category:Protocols]]

Yeast Genomic DNA - Revision history

Paul.xu at 23:05, 28 June 2014