Sorry, in my first answer I did not answer your question!

We always transform
bacteria with plasmids obtained from yeast, and then do miniprep. For yeast
plasmid extraction we use the following method:

Buffer A: 100 mM NaCl 10 mM Tris-HCl (pH 8) 1 mM EDTA 0.1 % SDS

1. Culture the plasmid-harboring cells overnight in 1 - 2 ml of medium. Cells with 1 -

4 OD600/ml are needed.
2. Pulse 5-10 sec at 15'000 rpm (maximum speed) (in an Eppendorf tube).
3. Throw away the supernatant.
4. Resuspend in 200 µl of buffer A, keep cells on ice.
5. Add glass beads just before the solution surface, mix with a vortex during 1
minute and sonicate.
6. Add 200 µl of phenol.
7. Vortex another minute.
8. Centrifuge at 15'000 rpm for 1 minute.
9. Throw away the phenol phase.
10. Add 200 µl of phenol and reextract the same way.
11. Take the water solution (about 200 µl) which contains the plasmids and treat
with Glassmilk: add 600 µl of NaI solution and 5 µl Glassmilk suspension, put the
tubes on the wheel for 5 min., then pellet the Glassmilk/DNA complex (pulse 5 sec),
remove the supernatant and set aside, then wash the pellet 3 times with 300 µl
New Wash, then pulse for 5 sec to remove the New Wash.
12. Elute the DNA into water (20 µl) or TE buffer.
When not using the GeneClean-kit, another protocol can be applied:Steps 1 to 9
remain the same as above. Then:
10. Add 200 ml phenol/chloroform 1:1 and vortex.
11. Centrifuge at 15'000 rpm for 1 minute.
12. Throw away the phenol phase.
13. Add others 200 µl of chloroform and reextract the same way.
14. Take the water phase and adjust the volume to 400 ml with water.
15. Add 40 ml 3M NaCl.
16. Add ca. 1 ml of ethanol 100 %.
17. Put the tube at -20 0C for about 10 min..
18. Centrifuge at 4 0C for 10 min. at maximum speed.
19. Throw away the supernatant.
20. Wash the pellet once with ethanol 80 % and once with ethanol 100 %.
21. Dry the pellet by putting the tube upside down on a Kleenex.
22. Resuspend the pellet in 50 ml TE buffer.
Transform bacteria with 20 µl.

Quick and Easy Isolation of Genomic DNA from Yeast

Author: Markus Ralser Source: Protocol Online Date Added: Mon Feb 02 2009 Date
Modified: Mon Feb 02 2009 Abstract: This protocol describes a quick and easy method for
genomic DNA preparation from yeast. The protocol can be used for PCR or southern blot
analysis
Procedure

1. Transfer 1.5 ml of liquid culture of yeast grown for 20 - 24 h at 30°C in YPD (1% yeast
extract, 2% peptone, 2% dextrose) into a microcentrifuge tube. Pellet cells by
centrifugation at 20,000 × g for 1-5 minutes.
2. Add 200 µl of Harju- buffer
3. Immerse tubes in a dry ice-ethanol bath for 2 minutes,
4. Transfer to in a 95°C water bath for 1 minute.
5. Repeat the last two steps
6. Vortex 30 seconds.
7. Add 200 µl of chloroform and vortex 2 minutes.
8. Centrifuge 3 minutes at room temperature, 20,000 × g.
9. Transfer the upper aqueous phase to a microcentrifuge tube containing 400 µl ice-cold
100% ethanol. Mix by inversion or gentle vortexing.
10. Incubate at room temperature, 5 minutes. Alternatively, precipitate DNA at -20°C to
increase yield.
11. Centrifuge 5 minutes at room temperature, 20,000 × g.
12. Remove the supernatant with a pulled Pasteur pipette by vacuum aspiration.
13. Wash the pellet with 0.5 ml 70% ethanol
14. Centrifuge 5 minutes at room temperature, 20,000 × g.
15. Remove supernatant.
16. Air-dry the pellets at room temperature or for 5 minutes at 60°C in a vacuum dryer.
17. Resuspend in 25- 50 µl TE (pH 8.0)] or water. Samples obtained directly from plates
should be resuspended in a 10 µl volume, because the yield will be smaller. 0.25 µl
RNase cocktail should be added to the samples used for Southern blot hybridization (final
concentration 0.125 U RNAse A, 5 U RNase T1).

Reagents

Harju- Buffer
– 2% Triton X-100
– 1% SDS,
– 100 mM NaCl
– 10 mM Tris-HCl, pH 8.0,
– 1 mM EDTA

Reference

Harju S, Fedosyuk H, Peterson KR. Rapid isolation of yeast genomic DNA: Bust n' Grab. BMC
Biotechnol. 2004 Apr 21;4:8.; PMID: 15102338