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| ChIP Protocol-Mechanical Breakage & FA Lysis Buffer
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| [ 文章来源： | 文章作者：
| 发布时间：2007-01-14|
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o Totals
1. Aliquot 300µl amounts of Chromatin Solution (no antibodies).
2. Add 3.2µl 5M NaCl.
3. Vortex.
4. Heat at 67&Mac251;C from 6 hours to overnight to reverse crosslinks.
Dave's Note: I usually prepare 2 total samples. That way, if something goes wrong during extractions, you don't have to start over from the beginning.
Pam's note: Might want to use lid locks or put heavy weight on top of all tubes to prevent evaporation.
Day Three
o Proteinase K Treatment
1. To IP's: Add 20µl 1M Tris-HCl pH 6.8 and 10µl 0.5M EDTA (make up cocktail and add 30µl to each tube)
2. To Totals: Add 33.3µl 10% SDS and 5.5µl 0.5M EDTA
3. Vortex.
4. Add 2µl Proteinase K solution.
5. Invert 3-4 times to mix.
6. Incubate @42&Mac251;C for 2 hours.
o Extractions
***For all extractions:
o Mix on TOMY mixer (max speed) for 2 minutes.
o Spin @10K, 5 min, room temperature. ***
1. Make phenol/chloroform/isoamyl alcohol (PCI) (25:24:1). Spin PCI @2K, 5min, RT, to make sure there is a water layer on top. Balance by weight (PCI is denser than water).
2. Extraction of IP's
o Extract once with equal volume PCI (500µl).
o Extract once with equal volume Chloroform (500µl).
o YYH recommends extracting with equal volume Chloroform (500µl) a second time.
3. Extraction of totals:
o Add 60µl TE.
o Extract twice with equal volume PCI (400µl).
o Extract twice with equal volume Chloroform (400µl).
o Back extract organic phases with 100µl TE.
For the totals extraction, set up five rows of tubes. Move the aqueous layer of the first PCI extraction to the second row. While extracting the second row with PCI, back extract the first row with TE. Again, move the aqueous layer from the first row to the second, from the second to the third, then extract the third row with chloroform and the second row with TE. Continue until all four extractions and back extractions are complete.
4. After extractions of both ChIP pellets and totals add:
5µg glycogen (from 20µg/µl stock - 0.25µl per tube)
50µl 3M NaOAc
1ml EtOH
Make a cocktail of glycogen and NaOAc, then add the EtOH separately.
5. Leave @-20&Mac251;C overnight.
Day Four
o Spin down all EtOH precipitates (ChIP and totals) @15K, 20min, 4&Mac251;C.
o Decant immediately after spin stops. Be careful because ChIP pellets should be VERY VERY TINY. If they are big it means there is SDS and they should be re-extracted with CHCl3 one more time and re-precipitated.
o Wash with 500µl 70% EtOH (DNA is not soluble in 70% EtOH).
o Spin @15K, 10min, 4&Mac251;C.
o Decant wash.
o Dry in speedvac.
o Resuspend in TE:
1. For ChIP pellets, we usually use 1µl/10 µl chromatin solution used for IP.
(For MIF2 and CSE4-HA under wild type conditions, can dilute in 0.5 µl/1 µl chromatin solution used for IP.)
amt used _________
2. For Totals, use 1µl/1 µl chromatin solution
amt used _________
Recipes
FA lysis buffer (500ml)
25 ml 1M HEPES-KOH, pH 7.5
14 ml 5M NaCl
1 ml 0.5M EDTA, pH 7.6
25 ml 20% Triton-x-100 (by vol)
10 ml 5% DOC (deoxychlolic acid)
425 ml sterile water
Add the following supplements to 10ml FA lysis buffer, when required:
10 µl 1mg/ml Leupeptin (Leu)
10 µl 1mg/ml Pepstatin (Pep)
50 µl 0.2M PMSF
Vortex immediately after adding PMSF.
FA Wash Buffer (500ml)
25 ml 1M HEPES-KOH, pH 7.5
50 ml 5M NaCl
1 ml 0.5M EDTA, pH 7.6
25 ml 20% Triton-x-100 (by vol)
10 ml 5% DOC (deoxychlolic acid)
389 ml sterile water
1X TE (100ml)
1 ml 1M Tris pH 8
200µl 0.5M EDTA pH 7.6
Fill to 100ml with sterile water.
LiCl/Detergent Wash (250ml)
2.65g LiCl
25 ml 10% NP-40
50 ml 5% DOC
0.5 ml 0.5M EDTA
2.5 ml 1M Tris pH 8.1
Alternative Breakage Buffer (probably less background with anti-HA)
SDS Lysis Buffer 100 mL:
1% SDS 10 mL 10% SDS (UltraPure)
10 mM EDTA 2 mL 0.5 M EDTA
50 mM TRIS, pH 8.1 5 mL 1 M TRIS
IP Dilution Buffer (for 1:9 dilution) 250 mL:
1.1% Triton X 100 13.8 mL 20% Triton X 100
1.2 mM EDTA 0.6 mL 0.5 M EDTA
16.7 mM TRIS, pH 8.1 4.2 mL 1 M TRIS, 8.1
167 mM NaCl 8.35 mL 5 M NaCl
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