Wednesday, February 25, 2009

Isolation of Yeast Genomic DNA

Isolating genomic DNA from yeast involves culturing the microbe, harvesting the cell, enzymatically removing the cell wall, lysing the protoplast, and finally separating the DNA from the other cell debris.
Materials that you need are:

Yeast culture-prepared previously

Spectrophotometer with cuvettes

50 mM EDTA, pH 8-ice cold

50 mM Tris, pH 9.5, 2% 2-mercaptoethanol

1.2 M sorbitol, 50 mM Tris, pH 7.5

Lyticase solution-500 U/ml in 50 mM Tris, pH 7.5

10% Sodium Dodecyl Sulfate (SDS)-used for checking protoplast formation

Lysis buffer-100 mM Tris, pH 7.5, 100 mM EDTA, 150 mMNaCl, 50 micrograms/ml RNase A

Lysis buffer with 2% SDS

95% Ethanol-stored at minus 20 degree Celcius

TE buffer-10 M Tris, pH 8, 1 mM EDTA

3 M potassium acetate, pH 5.5

Here are the step by step methods:

  1. The yeast can be cultured for as long as 48 hours at 30 degree Celcius. The optical density of a 1:10 dilution of the culture in water can be as high as 1.0 at 520 nm.
  2. Harvest 5 ml of cells by centrifugation (5 minutes at 5000 rpm). Resuspend the yeast in 1 ml of cold 50 mM EDTA, pH 8, and transfer to a 1.5 ml microfuge tube. Centrifuge for 1 minute, decant, and resuspend again in 50 mM EDTA.
  3. Pellet the cells as before and suspend the cells in 1 ml of 50 mM Tris, pH 9.5, 2% 2-mercaptoethanol. Incubate for 10 min at room temperature. Centrifuge and decant.
  4. Resuspend the cells in 800 micro liter of 1.2 M sorbitol, 50 mM Tris, pH 7.5. The sorbitol act as an osmotic support and prevents rupture of the cells as the wall is removed. As the yeast cell walls degrade, membranes can easily overextend and rupture.
  5. Add 200 micro liter of Lyticase (500 U/ml in 50 mM Tris, pH 7.5). Place the cells on a rocker and incubate at 37 degree Celcius for one hour. Lyticase is a yeast cell wall degrading enzyme isolated from the bacteria Arthrobacter luteus.
  6. Examine the suspension under a microscope to ensure protoplast formation. As the yeast wall is degraded, the cell membrane can ooze out of the sack. Viewed with phase contrast microscopy, yeast protoplasts are characteristically refractile (or bright) spheres, and yeast cell wall shells appear as gray ghosts (cell walls without membrane and cytosol). Combine 10 micro liter of 10% SDS with 10 micro liter of yeast protoplasts. Examine the cells under the microscope. The absence of refractile yeast indicates the protoplasts were lysed by the SDS.
  7. Pellet the protoplasts by centrifuging at 10000 rpm for five minutes. Resuspend the cells in 1 ml of 100 mM Tris, pH 7.5, 100 mM EDTA, 150 mM NaCl (lysis buffer). Transfer the cells to a 5 ml polypropylene tube. Add 1 ml of lysis buffer with 2% SDS. Mix and incubate at 30 Celcius egree for 30 minutes. Check the cells under a microscope for lysis.
  8. Centrifuge the lysate at 5000 rpm for 15 min to pellet cellular debris. Decant the upper phase containing the DNA.
  9. Using a pipet, determine the volume of the DNA solution. Add 1/10th volume (e.g., 100 micro liter for every ml) of 3 M potassium acetate to the solution. In the presence of ions Na and K, DNA precipitates if mixed with either ethanol or isopropanol. Incubate the DNA at -20 Celcius degree for 30 min (or overnight if possible. Centrifuge the solution at 7000 rpm for 20 minutes. The DNA appears as white pellet. Decant and remove as much moisture as possible, but do not allow the pellet to dry. Once genomic DNA drys, it can be very difficult to resuspend.
  10. Resuspend the DNA in 100 micro liter of TE buffer and freeze.

Reference: number 2 on References