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Help with cerevisiae DNA prep

Ricardo Azpiroz azpiroz at U.ARIZONA.EDU
Thu Jun 19 12:07:14 EST 1997

Here's a really nice prep for PCR, from the CSH yeast genetics course

Resuspend a glob of cells from a plate (about the size of a match head) in
50 microliters of water. Toothpicks work well for me. Boil five minutes.
Spin in microfuge 5-30 seconds. Use 5-15 microliters of supernatant as
template. Never failed me yet.


On Thu, 19 Jun 1997, Michael Lichten wrote:

In article <5o9aht$t2e$1 at usenet81.supernews.com>, meyerdj at phibred.com (Dr.
David J. Meyer) wrote:

> Southerns, and this has been working well. But although the protocol
> suggests that the DNA can be used for PCR, I find that it does not
> work for me using Taq (haven't tried anything else). It fails in a
> side-by-side comparison with DNA from a library pool.

Why do a DNA prep?

--PCR using DNA template from a yeast colony

1. Resuspend colonies: Use a pippet tip (NOT a toothpick) to pick up an
average-size yeast colony (not necessary to be fresh colonies of size
around 0.5-2 mm) and transfer to a tube containing 10 =B5l incubation
solution (1.2M sorbitol, 100 mM sodium phosphate, pH 7.4), resuspend cells
by pepitting up and down several times.

2. Spheroplasting cells: Add 1 =B5l 20 mg/ml zymolyase and mix. Incubate th=
tube at 37=B0C for 10 min.

3. PCR: Take 2~5 =B5l of the mixture to 30 =B5l PCR mixture containing prim=
and nucleotides. Heat the mixture at 94=B0C for 2 minutes, then 94=B0C 30 s=
Tm 30 sec, 72=B0C 45 sec for 30 cycles. Check 5 =B5l PCR reaction on a gel.

Ref: Ling et al. (1995) Nucleic Acids Research, 23:4924-4925.

Michael Lichten
lichten at helix.nih.gov

Ricardo Azpiroz
Department of Biochemistry
Life Sciences South
University of Arizona
Tucson AZ 85721-0106

azpiroz at U.Arizona.EDU

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