| Transformation of Yeast Spheroplast | 点击： 作者： 来源： 时间： 2007-04-11 本站论坛
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|  | Peter Novick Lab, Department of Cell Biology Yale University School of Medicine
http://info.med.yale.edu/cellbio/Novick/Second/Protocols/spheroplast.pdf
1. Grow a 100 ml YPD overnight culture of cells till the OD599=1.5-2.0
2. Spin down cells and wash once in sterile H2O.
3. Form spheroplasts at 37oC for 45 min in 20ml of spheroplast media.
4. Pellet the cells at 1100 rpm (~300 x g) in table top Beckman for 7 min.
5. Gently wash once in 1M sorbitol and pellet 5 min at 1100 rpm.
6. Gently resuspend in 20 ml of STC, and spin down cells for 5 min at 1100 rpm.
7. Gently resuspend in 2ml of STC. Aliquot 100µl of cells into 12 ml falcon tubes containing a
mixture of plasmid DNA and carrier DNA which totals 5 µg of DNA. I have used 0.2 µg of
plasmid DNA 4.8 µg of chick blood carrier DNA per tube and been very successful.
8. Incubate the tubes containing cells DNA for 10 min at room temperature.
9. Add 1 ml of PEG-8000 buffer to each tube and gently mix to resuspend.
10. Incubate at room temperature for another 10 min and then harvest for 5 min at 1100 rpm.
11. Resuspend in 150µl of SOS media supplemented with 10 µg/ml of selective amino acid (uracil
for example if using ura3-52 cells). Incubate at 30oC for 20-40 min. I have used a 40 min
incubation.
12. Add 7-8 mls of Top Agar, which is kept at 45-50oC, and invert tube to mix. The suspension
was quickly poured onto SORB plates and incubated at appropriate temperature. I have directly
placed the plates at restrictive temperature to select for complementing plasmids. Many colonies
that develop will be embedded in the agar.
13. For controls, include a no DNA sample and a tube containing plasmid but no insert. This will
control for revertants.
Stocks
STC media
1M sorbitol
10mM Tris-HCl, pH 7.5
10mM CaCl2
mix and filter sterilize
SOS media
1M sorbitol
6.5mM CaCl2
0.25% yeast extract
0.5% peptone
mix and filter sterilize
PEG-8000
20% PEG-8000
10mM Tris-HCl, pH 7.5
10mM CaCl2
mix and filter sterilize
Top Agar (for 200ml)
1M sorbitol
2.5% agar
2.68g/200ml of yeast nitrogen base w/o amino acids
mix and sterilize, then add 10ml of sterile 40% glucose
SORB Plates
13.4 g of Difco Yeast nitrogen base without amino acids
164 g sorbitol
1 liter of water
place in 2 liter flask and sterilize
40 g Bacto-agar
164 g sorbitol
1 liter of water
place in 4 liter flask with stir bar and sterilize
60 g glucose
100 ml of water
sterilize
When above are cool enough to touch, mix all in the 4 liter flask and stir on warm
setting till ~60oC. Pour some into a sterile liter flask and pour plates.
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