David Rose asks for a good yeast miniprep protocol.
Pek Lum, a graduate student in my lab, discovered that Magic
minipreps can be used to prepare plasmids from yeast. We really like
the purity of the DNA from these preps - it cuts, it transforms, etc.
And it doesn't appear contaminated with genomic.
Here is her protocol:
1. Grow up 10 ml saturated culture.
2. Harvest cells and resuspend in 1 ml of Zymolyase solution (2 mg
/ml 20T Zymolyase in 100mM KH2PO4 pH 7.5, 1.2 M sorbitol). Transfer
suspension to 1.5ml microcentirfuge tube and incubate at 37C for 1
hr. Check lysis by mixing 10 ul of cell suspension with 10 ul of the
Cell lysis solution from the Magic Miniprep kit. Observe by
microscopy, comparing with 10 ul of cell suspension that has not been
treated with Cell lysis solution. If more than 60% of the cells are
lysed, proceed. If not, continue incubation for 15 minutes and
recheck. Pek usually just digests for 2 hours without checking.
3. Add 200 ul of Cell Lysis Solution to the cell suspension.
Incubate at 65C for 5 min.
4. Add 200 ul of Neutralization Solution. Mix gently by inversion.
Chill on ice for 15 min.
5. Microcentrifuge at top speed for 5 min.
6. Transfer the clear supernatant solution to a fresh 1.5 ml tube.
Add 1 ml of the Purification Resin. Mix by inversion.
7. SUbsequent steps are performed using a vacuum manifold according
to Promega's instructions.
8. Elute DNA from the resin with 50 ul. For transformation into
bacteria, use 5-10 ul of the DNA.
9. NOTE: for S. pombe cells, Pek uses a mixture of 2mg/ml Zymolyase
and 2mg/ml Novazyme to digest the cell walls.
If you need more DNA, this protocol can be scaled up.