Peter Novick Lab, Department of Cell Biology Yale University School of Medicine
http://info.med.yale.edu/cellbio/Novick/second/protocols/lysis.pdf
Stocks
a. 10 mM Tris-HCL, pH 7.5, 10 mM NaN3
b. lysis buffer, 4oC (0.8 M sorbitol in 20 mM Triethanolamine, 1 mM
EDTA pH 7.2) containing 1 mM PMSF and 10 ul/10 ml protease inhibitor
cocktail: leupeptin, chymostatin, pepstatin, aprotinin, and antiparin 1
mg/ml.
Stocks
49.5 ml 0.8M SORB/TEA
25 ul 200 mM PMSF
50 ul PIC (@ 10ul/10 ml:
leupeptin
chymostatin
pepstatin
antipain
aprotinin)
=1mg/ml of each?
c. Spheroplast media (50 mM Tris pH7.5, 10 mM NaN3, 1.4 M sorbitol,
40 mM B-mercaptoethanol, 0.125 mg/ml Zymolase-100T)
stock
50mM Tris-HCL, pH 7.5 1M@
10 mM NaN3
1.4 M Sorbitol
40 mM beta mercaptoethanol
0.125mg/ml zymolase-100T
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 to get
rid of precipitate that will be there.
Protocol
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)
2
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:
Remove one good-looking colony from the plate and put it into
a sterile tube which contains YP liquid media plus 2% glucose. Put into
25oC oven for two days?. Place stationary culture into cold room. Good for
one month. As culture ages more cells are dead so more stationary culture
is required for inoculation of other cultures.
4. To prepare culture for experiment:
With a fresh stationary culture: 300 ul for 300 mls of YP plus
2%D. Shake in water bath at 25oC overnight (usually inoculate at 5:oo pm).
5. Next day determine O. D. of growing culture. Want near 1 O.D (200
OD599 Units) reading: 100ul of culture 900 ul of water; control 100 ul of
culture media, no bugs.
6. Spin culture in large plastic 100 ml tubes in Beckman GPR centrifuge
at 3,000 rpm for 5-10 min. at RT.
7. For a ts shift:
Have prepared a flask with 300 mls of YP 0.1% glucose,
warmed to 37oC. (Can set this up night before when inoculating for the
experiment. Serves two functions: 1: Warms media; 2: Tests for
contamination. Resuspend pellet into this media. Shake at 37oC for 40 min
to 1 hr.
8. Spin down culture as in step 6. Wash one time with Tris/HCL pH 7.5
with 10 mM Na N3 (NaN3 not appropriate in all applications) RT.
9. Resuspend cells in 30 mls spheroplast media. Transfer to a tube that
allows centrifugation under correct conditions: tube # . Incubate 45
min at 37oC. Flip the tube gently ever 5-10 min during incubation. Cool
mixture after incubation on ice for a few min. Pellet 5 min on high using
the Dynac centrifuge.
10. Resuspend cooled pellet in cold lysis buffer. Pellet will be soft; pour
supernatant gently out of tube by hand, then use pipette to remove last
3
drop. Amount buffer added depends on the concentration of lysed cell
stuff desired. Do two lysis steps with 3-4 mls at each step as follows:
following steps were performed at 4oC .
a. The suspension was homogenized 20 times in a 40 ml Wheaton tissue
grinder, pestle A, and centrifuged at 450 xg for 3 min. Use the
centrifuge 1900 rpm for 3 min. Need to precool centrifuge and rotor.
b. The pellet (P1) was resuspended in the same volume lysis buffer,
homogenized and centrifuged as above, and the supernatants (S1) pooled.
c. Spin S1 supernatant at 10,000 xg for 10 min. Use SS-34 tubes for 10
min in the Beckman J2-21.
d. The supernatant (S2) was spun at 100,000 xg for 1 hour to produce a
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