Peter Novick Lab, Department of Cell Biology Yale University School of Medicine
http://info.med.yale.edu/cellbio/Novick/second/protocols/growth.pdf
Cell Growth, Lysis and Fractionation
I . Stocks
a. Wash Buffer: Final concentration: 10 mM Tris-HCL, pH 7.5, (10 mM NaN3 if
applicable); pH stock with concentrated (12 M) HCL. Also check pH of 1X solution.
100 mls 200ml 500 mls Stock Stock Concentr ation
1 ml 2 ml 5 mls Tris-HCL, pH 7.5 1 M
99 ml 198 ml 485 mls dH2O (
c. Spheroplast Media (Final concentration: 50 mM Tris pH7.5, (10 mM NaN3 if
applicable), 1.4 M sorbitol, 40 mM B-mercaptoethanol, 0.125 mg/ml Zymolase-100T)
30 mls 60 mls 90 ml Stock Stock Concentration
1.5 ml 3 mls 4.5 ml Tris-HCL, pH 7.5 1 M
-------- -------- --------- NaN3 0.5 M
10.5 mls 21 mls 31.5 ml Sorbitol 4.0 M
120 ul 240 ul 360 ul beta mercaptoethanol 100%= 13.2M
0.0038 grm 0.0075 grm 0.01125 gr zymolase-100T powder, kept 4oC
17.88 ml 35.76 ml 53.64 ml ddH2O
Make solution up at least 20 min ahead of time cause zymolase is a bear to get into solution.
Shake etc. but after 20 min pellet solution (Dynac centrifuge, on High, 3 min) to get rid of
precipitate that will be there, but not visible (want to remove any theoretical protease cause
don't want protease in lysate).
b. Lysis Buffer, 4oC (Final concentration: 0.8 M sorbitol in 10 mM Triethanolamine, 1
mM EDTA pH 7.2) containing 1 mM PMSF and 10 ug/1 ml protease inhibitor cocktail (PIC):
leupeptin, chymostatin, pepstatin, aprotinin, and antiparin )
5mls 10 mls Stock Stock Concentration
1 mls 2 mls Sorbital 4.0 M
0.5 mls 1 mls TEAE 10X
25 ul 50 ulsPMSF 100 mM (-20oC)
1 ul 5 ul PIC 1 mg/ml of each: (-20oC stock)
leupeptin
chymostatin
pepstatin
antipain
aprotinin
3.47 mls 6.95 mls dH2O
NOTE: PMSF goes bad in 30 min on ice so replentish if expired or add just before use. Also
apply same expiration criteria for PIC
d. YP sterile liquid (Kim makes it as a lab stock)
Heike has a recipe in her 5x8 inch file box.
e. 40% glucose, sterile liquid (Kim)
40 grams in 100 mls
f. Sorbital, 4M
MW= 182.2; For 500 mls in H2O= 364.4 grams, Microwave on High 5 min; Cool;
Bring to volume.
g. TEAE, 10X
Final concentration of 10X Stock: 10 mM EDTA, 100 mM TEA (triethanolamine), pH to
7.2 with with glacial acetic acid.
500 mls Stock
7.45 grams TEA liquid, FW 149.2 grms/mole: weigh out in a test tube.
375.24 grms EDTA crystals, FW 372.24 grams/mole
H2O to volume
II. Procedure
1. Frozen strain stocks were streaked onto a YPD plate (=1% yeast extract, 2% peptone, 2%
dextrose). Take sterile wooden sticks, labelled YPD plate and gloves up to freezer. Remove yeast
from freezer vial with wooden stick by gouging out a chunk of frozen stuff. Smear onto plate in
one small place. Replace freezer vial. Go downstairs to lab. Remove sterile 1 ml glass pipette.
Flame tip. Cool by placing pipette tip into agar. Streak yeast out in the following pattern and be
sure to reach into the original smear while streaking for the first part of the pattern: (goal is to get
single colonies)
(SEE LAST PAGES FOR DIAGRAM)
2. Put plate upside down into 25oC oven in Susan's lab for two days. Remove plate and put
into 4oC refrigerator.
3. Make a stationary culture from the plate:
a. Remove one good-looking colony from the plate with a sterile, flamed, cooled 1 mls pipette (or
loop). Just swipe at the colony.
b. Put pipette into a sterile tube which contains 3mls or 5mls ofYP liquid media plus 2% glucose.
Stir the pipette around a little. Flame top of tube.
c. Put tube into 25oC water shaker bath setting 6 for shaking. Be sure to leave it shaking! Leave
for three days. Place stationary culture into cold room. Good for one month if in YPD
media. As culture ages, more cells are dead; therefore, more volume of the stationary
culture is required for inoculation of other experimental cultures.
4. To prepare culture for experiment:
With a fresh stationary culture: 300 ul for 300 mls of YP plus 2%D. Place a check on label of
culture after adding glucose. Shake in water bath at 25oC overnight (usually inoculate at 5:00 pm).
Doubling time for YPD and wild-type cells is 2 hours.
5. Next day determine O.D. of growing culture. Want near 1-2 O.D (200 OD599 Units).
Closest to 1= O.D. is best.
Reading: 100ul of culture 900 ul of water
Control: 100 ul of culture media, no bugs 900 ul of water.
6. Spin culture in large plastic 250 ml or 100 ml tubes in Beckman GPR centrifuge (The
Benchtop) at 3,000 rpm for 5-10 min. at RT.
7. For a ts shift:
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