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Help with cerevisiae DNA prep

Michael Lichten lichten at helix.nih.gov
Thu Jun 19 09:54:40 EST 1997


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 µl 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 µl 20 mg/ml zymolyase and mix. Incubate the
tube at 37°C for 10 min.

3. PCR: Take 2~5 µl of the mixture to 30 µl PCR mixture containing primers
and nucleotides. Heat the mixture at 94°C for 2 minutes, then 94°C 30 sec,
Tm 30 sec, 72°C 45 sec for 30 cycles. Check 5 µl PCR reaction on a gel.

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

-- 
Michael Lichten
lichten at helix.nih.gov



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