Chromatin Isolation
From Genome@Yale
Solutions
Lysis Buffer 1 (L1). Add 1 protease inhibitor tablet (Roche Complete cat #1 697 498).
Final Stock Volume for 60mL 2X 50mM Hepes pH 7.5 1M 3mL 140mM NaCl 5M 1.68mL 1mM EDTA 0.5M 120uL 10% Glycerol 100% 6mL 0.5% NP-40 10% 3mL 0.25% Triton X-100 10% 1.5mL dH2O 100% to 60mL
Buffer 2 (L2, Protein Extraction Buffer) Kept at RT. Add 1 protease inhibitor tablet per 50mL.
Final Stock Volume for 50mL 2X 200mM NaCl 5M 2mL 1mM EDTA pH8 0.5M 100uL 0.5mM EGTA 0.5M 50uL 10mM Tris, pH8 1M 500uL dH2O 100% 47.35mL
Buffer 3 (L3, Chromatin Extraction Buffer) Kept on ICE. Add 1 protease inhibitor tablets per 50mL.
Final Stock 10mL Vol for 20mL 1mM EDTA pH 8 0.5M 20uL 40uL 0.5mM EGTA pH 8 0.5M 10uL 20uL 10mM Tris pH 8 1M 100uL 200uL Complete (Roche) 50X 200uL 400uL dH2O 100% 9.67mL 19.34mL
- Thaw cell pellets in ice water (takes 1hour or longer). Resuspend cells in 10mL of buffer L1. Rock at 4°C for 10 minutes.
- Spin at 3000 rpm in 10 minutes at 4 °C.
- Resuspend each tube of cells in 10mL of buffer L2.
- Rock at room temperature for 10 minutes.
- Pellet nuclei in a tabletop centrifuge by spinning at 4K rpm, 10 minutes at 4°C.
- Resuspend nuclei in 5 mL buffer L3. Transfer to a 15mL conical tube.
- Sonicate the cell suspension with a microtip attached to Branson 450 sonifier, setting at 4 (about 25% power output), constant power (place the 15mL conical tube in a 50mL conical tube with ice).
- Sonicate 8 times for 20 seconds, allowing the suspension to cool on ice between pulses for 40 seconds.
- Spin out debris at 4K rpm, 15 minutes.
- OD samples
- Adjust the final glycerol concentration to 10% with 80% glycerol stock solution.
- Adjust the final DNA concentrations to 2mg/mL by adding L3 buffer.
- Freeze chromatin at -80°C in 2mg aliquots (1mL).
- Chromatin samples stored at -80°C.
[edit]
Analyze sonicated chromatin
Proteinase K - RNase Mix
Proteinase K Mix
1X Sample 10X 13X 141uL TE 1.41mL 1.823mL 1uL Glycogen 20mg/mL 10uL 13uL 7uL Proteinase K 20mg/mL 70uL 91uL 1uL RNase A 10mg/mL 10uL 13uL
- incubate 50uL of chromatin with 120uL of Elution Buffer (TE + 1% SDS) at 65°C O/N
- add 150uL Proteinase K - RNase Mix and incubate at 37°C 2hours
- add 300uL phenol:chloroform:isoamyl alcohol
- transfer aqueous phase to a new tube and add 13 uL 5M NaCl and 750uL 100% EtOH
- freeze for 15 minutes at -80°C
- spin at 15K rpm 15 minute 4°C
- wash pellet with 70% EtOH
- air dry pellet and resuspend in 50uL dH2O
- analyze fragments on 1% agarose gel
