New ChIP Protocol (3/25/11) Adjusted
The protocol ideally requires 100mg (≈100µl embryos) of starting sample. Day 1 can be repeated as needed to
stockpile enough starting sample to proceed. Embryo processing can take ≈30 min. prior to fixing, so schedule
aging times accordingly.
Day 1: Sample Prep, Fix, and DNA Shearing
**Protease Arrest should be brought to RT and added to the Nuclear Isolation Buffer to 1x conc. (1µl PA in
100µl NIB)
PA must also be added to the RIPA NS ΔEDTA Buffer to 2x conc. (5µl PA in 250µl RIPA NS buffer)
Also, ice cold PBS is needed.
If planning to proceed to Day 2 of protocol the same day, allow ample time to aliquot and prewash beads. Prewash beads by adding 1mL regular RIPA buffer, rotating briefly, spinning down @ 700g and removing sup.
After sonication, barrier tips should be used for the rest of the protocol.
1) Collect, dechorionate, and wash embryos with PBST, then put embryos in 1.5mL tube.
2) Add 100µl Nuclear Isolation Buffer+Protease Arrest. Grind thoroughly with motorized homogenizer
≈1 min.
3) Add additional 100µl NIB+PA.
4) Spin 6 min. RT @500g. Carefully transfer sup to new 1.5mL tube. (The extract may still be
homogeneous after the spin but should still be transferred to a new tube to separate it from any
remaining large cellular debris on the sides of the old tube.)
5) Add Paraformaldehyde to 1% final concentration. (≈25µl of 10% Paraformaldehyde) Rotate 15 min.
RT, flicking as needed to ensure proper mixing.
6) Add Glycine to 0.25M final concentration. (≈92µl of 1M Glycine) Spin 6 min RT @3000g.
7) Remove and discard sup. Gently add 500µl ice cold PBS to the pellet, remove sup and cellular debris.
REPEAT 2x for a TOTAL of 3. (Using a cut tip may help in removing larger debris in the sup. Even so,
at the end of wash 3 there may still be some debris-this is ok.)
8) Add appropriate volume of RIPA NS ΔEDTA Buffer+Protease Arrest to the pellet (see table below).
Pipet, flick, and vortex as needed to dislodge the pellet and resuspend it into solution. (There may still
be large pellet pieces that will not completely dissolve-this is ok.) Pop spin to get any pellet pieces from
the side of the tube into the buffer. Put samples on ice and proceed to sonication.
Starting Tissue Amount RIPA NS ΔEDTA Volume
<75 µl
200 µl
75-100 µl
240 µl
9) Sonication: Power should be set to 3. See table below for appropriate pulse guideline. Samples should be
allowed at least 1 minute on ice between pulses. Also, cool the sonicator tip with ice cold water and
sterilize with alcohol between pulses if switching genotype. (If the extract becomes too hot during a 40
second pulse, it is ok to pulse for 20 seconds, ice for ~5 seconds, and then pulse for 20 more seconds).
Samples should remain on ice when finished sonicating.
Pulse guideline
7 pulses, 4 @30 sec. and 3 @ 40 sec.
Use Barrier Tips Beyond This Point:
10) Verify Shearing (Majority of DNA fragment size should be 200-700bp): Run 1.6% of extract(3.3µl of
200µl or 4µl of 240µl)+3µl loading dye for 20 min on a 1% agarose gel WITHOUT EtBr. Stain and
destain 10 min. each. Re-sonicate if needed.
11) Once optimal shearing is confirmed, add Triton to 1% final concentration (see table below). Nutate 1
min. RT
12) Add Na-deoxycholate to 0.1% final concentration (see table below). Nutate 1 min. RT
13) Add NaCl to 140mM (see table below). ROTATE 5 min. in the COLD ROOM.
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RIPA NS ΔEDTA Triton (10% stock)
Na-deoxycholate (10% stock) NaCl (5M stock)
200 µl
23 µl
2.3 µl
6.5 µl
240 µl
29.6 µl
2.96 µl
8.32 µl
14) Spin 10 min 4ºC @10,000g. Transfer sup to new 1.5mL tube and discard pellet.
15) Write approximate starting sample amount on tube (i.e. 100mg (≈100µl embryos)) to make combining
extracts for the IP easier.
If enough sample for the IP was collected (≈100mg), proceed to Day 2 of protocol. Otherwise snap freeze
on dry ice and store @ -80ºC
Day 2: IP
**Aliquoting and prewashing beads should be done first. Use barrier tips.
16) Combine respective extracts and add to appropriate prewashed beads to pre-clear. Use 20µl of beads
per extract. (Ex: For 2 combined extracts, use 40µl of beads). Rotate 1 hr. RT.
17) Spin 30 seconds RT @ 700g. Transfer sup to new 1.5mL tube.
18) Take Input sample, store @ 4ºC in 1.5mL tube. (This is 10% of final extract volume: i.e. 35µl input for
350µl extract volume after input is taken. Can multiply starting amount by 0.090909 to obtain 10% of
final extract value.)
19) Bring volumes up to 600µl using RIPA buffer (volume can vary depending on antibody strength and
background levels)
20) Add Antibody to extract. (Amount varies by antibody: i.e. 2.0µl for αH3K9me2)
21) Nutate 1 hr RT and O.N. in the cold room.
Day 3: Finish IP, Wash, and Reverse Crosslinks
**Aliquoting and prewashing beads should be done first. Frozen Proteinase K should be thawed when washes
are close to finishing. Use barrier tips.
22) Pop spin Ab bound extract.
23) Add extract to 30µl prewashed beads.
24) Rotate 1.5 hrs. in the cold room and 1.5 hrs RT.
25) Spin 30 seconds RT @ 700g. Save sup (lysate) in 1.5mL tube.
Washes:
26) Add 1mL RIPA Buffer to beads. Rotate 10 min. RT. Spin 30 seconds RT @ 700g. Discard sup.
27) Add 1mL Wash Buffer 2 to beads. Rotate 10 min. RT. Spin 30 seconds RT @ 700g. Discard sup.
REPEAT 1x for a TOTAL of 2 washes.
28) Add 1mL Wash Buffer 3 to beads. Rotate 10 min. RT. Spin 30 seconds RT @ 700g. Discard sup.
REPEAT 3x for a TOTAL of 4 washes.
29) Add 1mL Wash Buffer 4 to beads. Rotate 10 min. RT. Spin 30 seconds RT @ 700g. Discard sup.
REPEAT 1x for a TOTAL of 2 washes.
30) Add 1mL Wash Buffer 5 to beads. Rotate 10 min. RT. Spin 30 seconds RT @ 700g. Discard sup.
REPEAT 1x for a TOTAL of 2 washes.
31) Once the last wash sup is discarded, add 500µl TE+0.5% SDS to beads. Also, remove Input sample
from the fridge and add TE+0.5%SDS to 500µl total volume. From now on, everything that is
done to the IP sample must also be done to the Input sample exactly. Add 10µl Proteinase K
(20mg/mL stock). Mix and incubate 37ºC 1 hr.
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32) Add SDS to 1% final concentration (≈60µl of 10% SDS) and NaCl to 0.3M final concentration (≈36µl
of 5M NaCl). Mix.
33) Incubate 65ºC O.N.
Day 4: Phenol/Choloroform Extraction and PCR
**50:50 Neutral Phenol:Choloroform, TE, NaOAC pH 5.2, glycogen, ice cold 100% EtOH, and ice cold
70% EtOH are needed. Use barrier tips.
34) Vortex and spin 30 seconds 1500g RT. Transfer sup off of beads to a new 1.5mL tube for extraction.
35) Add 500µl Neutral Phenol:Chloroform (50/50)
36) Vortex to mix. Spin 5min at max at RT.
37) Transfer aqueous layer to new 1.5mL tube. Back extract organic layer by adding 50µl of TE to the
organic layer, vortexing and spinning 5min at max at RT, and combining the resulting aqueous layer to
the one already collected.
38) Add another 500µl Neutral Phenol:Chloroform (50/50) to combined aqueous layer.
39) Vortex to mix. Spin 5min at max at RT.
40) Transfer aqueous layer to 2mL tube. Back extract organic layer again by adding 50µl of TE to the
organic layer, vortexing and spinning 5min at max at RT, and combining the resulting aqueous layer to
the one already collected in the 2mL tube.
41) 3M NaOAc pH 5.2 (25µl), and 1µl glycogen. Mix well. Add 2 volumes of ice cold 100% EtOH
(≈1.2mL), top off with 100% EtOH if possible. Mix well.
42) Place in -20oC for 30 min.
43) Spin tubes 15min at 4oC at max speed.
44) Carefully remove supernatant. Gently rinse pellet with 500µl ice cold 70% EtOH and spin 5-7 min at
max speed at 4oC. Carefully discard sup
45) Vacuum dry 5-20 min as needed and resuspend pellet in 30µl Millipore water.
46) Proceed to PCR or store at 4oC for short term(≈1 week)/ -80ºC for long term.
47) PCR:
**Make 1:10 dilution of the Input DNA only. The Input will now be 1% of total extract. The IP DNA
will remain undiluted.
Use appropriate primer sets, GoTaq 2x MM, and water to set up 20µl reactions (18µl of MM and 2µl of
DNA. Use the 1:10 dilution Input DNA for the PCR.
Run program: ChIP33 (change annealing temps and extension times as needed for primer sets)
Visualize products on 1.2% Agarose Gel WITH EtBr.
Run additional cycles (program ChIP4) as needed.
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ChIP Solutions
Nuclear Isolation Buffer (NIB)
100mL
50mM Hepes pH 7.6
10mL of 0.5M stock
60mM KCl
6mL of 1M stock
250mM Sucrose
25mL of 1M stock
Protease Inhibitor *(add on day of use)
1:50
RIPA NS ΔEDTA Buffer
100mL
0.1% SDS
1mL of 10% stock
10mM Tris pH 8.0
1mL of 1M stock
10mM EDTA pH 8.0
2mL of 0.5M stock
Protease Inhibitor *(add on day of use)
1:7
RIPA Buffer
100mL
1% Triton X-100
10mL of 10% stock
1mM EDTA
200µl of 0.5M stock
0.5mM EGTA
100µl of 0.5M stock
10mM Hepes pH 7.6
2mL of 0.5M stock
0.1% Na-Deoxycholate
0.1g (or 1mL of 10% stock)
0.1% SDS
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140mM NaCl
5.6mL of 2.5M stock
ChIP Solutions Cont’d.
Wash Buffer 2
200mL
50mM Tris pH 8.0
10mL of 1M stock
2mM EDTA
800µl of 0.5M stock
800mM NaCl
64mL of 2.5M stock
1% NP40
20mL of 10% stock
Wash Buffer 3
200mL
50mM Tris pH 8.0
10mL of 1M stock
2mM EDTA
800µl of 0.5M stock
500mM NaCl
40mL of 2.5M stock
1% NP40
20mL of 10% stock
0.1% SDS
2mL of 10% stock
Wash Buffer 4
200mL
100mM Tris pH 8.0
20mL of 1M stock
2mM EDTA
800µl of 0.5M stock
1% NP40
20mL of 10% stock
500mM LiCl
25mL of 4M stock
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1% Na-Deoxycholate
2g (or 20mL of 10% stock)
ChIP Solutions Cont’d.
Wash Buffer 5
200mL
10mM Tris pH 8.0
2mL of 1M stock
0.5mM EDTA
200µl of 0.5M stock
150mM NaCl
12mL of 2.5M stock
0.1% Tween 20
2mL of 10% stock
2mM DTT
400µl of 1M stock
TE+0.5% SDS
10mL
0.5% SDS
TE
500µl of 10% stock
9.5mL
Egg Fix 5x Buffer
100mL
250mM Hepes PH 8.0
50mL of 0.5M stock
5mM EDTA
1mL of 0.5M stock
500mM NaCl
20mL of 2.5M stock
Egg Fix Buffer+10% Paraformaldehyde (make fresh every other day, store a 4ºC)
2.5mL
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1x Egg Fix Buffer
0.5mL of Egg Fix 5x Buffer
2mL Millipore Water
10% Paraformaldehyde
0.25g
*Heat Egg Fix 1x Buffer to the brink of boiling to dissolve solid Paraformaldehyde. Cool by running under cold
water
Neutral Phenol: Add 1M Tris pH 8 to Phenol, mix, and allow to equilibrate. Remove aqueous layer, add
another round of 1M Tris pH 8 to Phenol layer, mix, and allow to equilibrate. Take pH. It should be around 8.
Remove aqueous layer, add 10mM Tris pH 8, mix, and allow to equilibrate. Store in the fridge.
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