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Chromatin Preparation
Two days before prep, inoculate 5ml media (YP + 2% Raffinose). 30 ºC 24 hrs.
Day before prep, inoculate 300 ml media (YP + 2% Raffinose) with overnight culture.
For YP-Raffinose, 300 ul wt will reach OD600=0.4 in ~16 hours.
Day 1
For galactose induction, add 15 ml 40% galactose at OD=0.4. Grow one hour.
At OD=0.5, add 1/10 volume of newly made Fix solution.
Shake slowly at RT for 5-60 minutes.
- For Flag-Ste12, I crosslinked for 60 minutes
Add 18 ml 2.5 M Glycine per 100 mL culture
Shake at RT for 5 minutes
Pellet cells (GS3, 5 min, 6000g)
wash twice with 10 ml ice cold TBS in 15 ml screwcap tubes
- if necessary, cells can be frozen at this step.
Resuspend cells in 500 ul Breaking Buffer + 1 mM PMSF
transfer to flat bottom 2 ml microcentrifuge tube
add ~500 ul glass beads
vortex in cold room, full speed, for 40 minutes
With 20 gauge needle, puncture bottom and then top of tube.
place tube immediately into 15 ml screwcap tube
spin 1000 rpm, 2 min in tabletop centrifuge
Add 1 ml FA buffer + protease inhibitors
transfer to 2 ml flat bottom tube
14k 1 min 4ºC
remove supernatant carefully with pipette
Wash pellet twice with 1 ml FA buffer + PIs
(you should resuspend pellet by gentle pipetting)
Resuspend chromatin pellet in 300 ul FA + PI's
sonicate with microtip: 12x 10 sec at 1.5 setting with 2 minute breaks on ice between each round of sonication
Add 1.2 ml FA + PIs and mix gently
spin 14 k, 30 min, 4 ºC
turn tubes and repeat 30 minute spin. (this gives you tight pellet)
Save 20 ul to check shearing and freeze rest of supernatant (chromatin sample) by freezing in liq. N2 and storing at -70.
Add 70 ul H2O and 90 ul 2x Stop Buffer to 20 ul of chromatin.
add 1 ul (20 micrograms/microliter) glycogen
Incubate at 75 ºC (in hybridization oven) overnight.
Day 2
Add 20 micrograms proteinase K to chromatin
incubate at 55 ºC (hybridization oven) for at least three hours.
Phenol extract once
Phenol/chloroform extract once
EtOH precipitate. Wash once with 80% ethanol and dry briefly in speedvac.
Resuspend in 30 ul TE + 10 micrograms RNase A
37 ºC 1 hr.
Add 20 ul TE and purify with invitrogen PCR cleanup kit. Elute in 50 ul elution buffer.
Run 15 ul on agarose gel to check shearing. Should be centered around 300-600 bp.
Immunoprecipitation
Prepare beads - Day before immunoprecipitation
make 7 ml of 5 mg/ml IgG-free BSA in PBS fresh
Pipette 20 ul magnetic Protein G beads per IP into siliconized eppie.
(make at least 80 ul to ensure proper mixing during overnight incubation)
Wash with 3 times with 1 ml BSA solution - rotate on nutator for 5 min at RT each time
Add 100 ul/IP of BSA solution and resuspend well
add 1 ul/IP of anti-Flag M2 antibody (~4 mg/ul)
Rotate overnight at 4 ºC (usually use Toshi lab's rotator)
Immunoprecipitation
Wash beads twice with 1 ml BSA solution as above
resuspend in 30 ul/IP of BSA solution and keep on ice
Thaw chromatin quickly in RT water bath and aliquot amount needed
spin 14k 15 minutes 4ºC
- for a single IP, I usually take 250 ul (200 for IP and 20 ul for input DNA and, if desired, western analysis)
- refreeze rest of chromatin in liquid nitrogen
Filter chromatin:
prewet Millipore Ultrafree-MC 0.45 ºm filter unit with 50 ul FA buffer
spin 14k 1 min 4ºC and discard wash
apply chromatin to filter and spin 14k 1 min 4ºC
save 20 ul for input DNA and, if desired) 20 ul for western blotting
Add 200 ul filtered chromatin to 30 ul Dynabeads
rotate 90 minutes at room temperature
Wash beads (1 ml buffer, 5 minutes on nutator, room temperature)
3 times with FA
2 times with FA-HS (high salt)
1 time with RIPA
Elute:
resuspend beads in 10 ul RIPA containing 3x-Flag peptide (5 mg/ml)
add 40 ul RIPA
30 min. room temperature. 1200 rpm in thermomixer
repeat elution and combine.
if desired, save 10 ul for western blot
continue with DNA preparation or freeze in liquid nitrogen
DNA preparation:
add equal volume (90 ul) of 2x stop buffer to eluted DNA.
add 70 ul H20 and 90 ul 2x stop buffer to input DNA.
add 1 ul glycogen (20 ug/ºl) to all samples
incubate at 75 ºC overnight in hybridization oven.
add 20 ug proteinase K (self-digested)
incubate at 55 ºC for at least three hours.
{alternatively, can incubate at 75 ºC for 6 hours and 55 ºC overnight}
Phenol extract once
Phenol/chloroform extract once
EtOH precipitate, wash once with 80% ethanol and dry briefly in speedvac
Resuspend in 30 ul TE and 10 ug RNase A
incubate 37 ºC for 1 hour
Add 20 ul TE and purify using invitrogen PCR cleanup kit
elute in 50 ul kit elution buffer.
Reagents:
Fix Solution (make fresh on day of crosslinking) 30 ml
11 % formaldehyde 8.9 ml 37 % Formaldehyde
100 mM NaCl 0.6 ml 5 M NaCl
1 mM EDTA 0.12 ml 0.25 M EDTA
50 mM Hepes pH 7.6 1.5 ml 1 M Hepes pH 7.6
2.5 M Glycine
TBS 500 ml
20 mM Tris pH 7.6 10 ml 1 M Tris pH 7.6
150 mM NaCl 15 ml 5 M NaCl
Breaking Buffer 2.5 ml
20 mM Tris pH 8.0 0.25 ml 1 M Tris pH 8.0
150 mM NaCl 1 ml 50 % glycerol
- add 1 mM PMSF fresh on day of chromatin prep.
FA Buffer 500 ml
50 mM Hepes pH 7.6 25 ml 1 M Hepes pH 7.6
150 mM NaCl 15 ml 5 M NaCl
1 mM EDTA 2 ml 0.25 M EDTA
1 % TritonX-100 5 ml 100 % TritonX-100
0.1 % sodium deoxycholate 0.5 g sodium deoxycholate
FA-HS Buffer 500 ml
as above, except add 50 ml of 5 M NaCl instead of 15 ml.
RIPA Buffer 500 ml
10 mM Tris pH 8.0 5 ml 1 M Tris pH 8.0
250 mM LiCl 25 ml 1 M LiCl
1 mM EDTA 2 ml 0.25 M EDTA
0.5 % NP-40 2.5 ml 100 % NP-40
0.5 % sodium deoxycholate 2.5 g sodium deoxycholate
2x Stop buffer 50 ml
20 mM Tris pH 8.0 1 ml 1 M Tris pH 8.0
100 mM NaCl 1 ml 5 M Nacl
20 mM EDTA 4 ml 0.25 M EDTA
1 % SDS 2.5 ml 20 % SDS
PCR:
For positive control, use a serial dilution of “input”. Exact dilution series will depend upon your IP signal strength. A reasonable starting series is 1:50, 1:250, 1:750.
For bound (IP) use undiluted DNA.
For primers: aim for PCR products around 200 - 350 bps and ann. temp. near 55 ºC
Reaction (20 ul)
2 ul 10x PCR buffer
1.6 ul 25 mM MgCl2
20 pmol primer 1
20 pmol primer 2
0.4 ul 10mM dNTPs
0.4 ul DMSO
0.4 ul Platinum Taq (or similar heat activated polymerase)
2 ul DNA sample
H2O to 20 ºl
PCR protocol
95 ºC 2 min - [95 ºC 20 sec - 55 ºC 40 sec - 72 ºC 30 sec]23-25 cycles - 72 ºC 5 min.
Cold PCR: exactly as above and run reaction on 2 % agarose
Hot PCR (necessary for quantitation):
add 0.1 ul [±-P32] CTP. (10 mCi/ml) to each reaction
after reaction, add 4 ul 5x DNA loading buffer (same as for agarose gels)
run half of sample on 6% Acrylamide gel with 1x TBE running buffer
6% Acrylamide gel
26.25 ml H2O
7 ml 30%/0.8% Acrylamide
1.75 ml 10x TBE
0.35 ml 10% APS
0.028 ml TEMED
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