ChIP

This protocol is in development.12:14, 21 August 2006 (EDT)

Overview

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Materials

11% Formaldehyde Solution

per 50 ml: (Final concentration)

• 14.9 ml of 37% Formaldehyde (11%)
  
• 1 ml of 5M NaCl (0.1M)
  
• 100 µl of 0.5 M EDTA, pH 8 (1 mM)
  
• 50 µl of 0.5 M EGTA, pH 8 (0.5 mM)
  
• 2.5 ml of 1M Hepes, pH 8 (50 mM)
  

2.5 M glycine (187.5 g/L)

• Dissolve glycine in water with constant stirring
  
• Don’t adjust pH
  

BSA/PBS Solution

per 100 mL:

• 10 mL of 10x PBS
  
• 500 mg of BSA
  
• 90 mL of H20
  

(good for at least a week)

Lysis Buffer 1 (LB1)

per 100 ml: (final concentration)

• 5 ml of 1M Hepes-KOH, pH 7.5 (50 mM)
  
• 2.8 ml of 5M NaCl (140 mM)
  
• 0.2 ml of 0.5M EDTA (1 mM)
  
• 10 ml of glycerol (10%)
  
• 5 ml of 10% NP-40 (0.5%)
  
• 0.25 ml of Triton X-100 (0.25%)
  

Lysis Buffer 2 (LB2)

per 100 ml: (final concentration)

• 4 ml of 5M NaCl (200 mM)
  
• 0.2 ml of 0.5M EDTA (1 mM)
  
• 0.1 ml of 0.5M EGTA (0.5 mM)
  
• 1.0 ml of 1M Tris pH 7.5 (10 mM)
  

Lysis Buffer 3 (LB3)

per 100 ml: (final concentration)

• 0.2 ml of 0.5M EDTA (1 mM)
  
• 0.1 ml of 0.5M EGTA (0.5 mM)
  
• 1.0 ml of 1M Tris-HCl, pH7.5 (10 mM)
  
• 2 mL of 5M NaCl (100 mM)
  
• 1 mL of 10% Na-Deoxycholate (0.1%)
  
• 500 mg of N-lauroyl sarcosine (0.5%)
  

Wash buffer (RIPA buffer)

per 100ml: (final concentration)

• 5ml of 1M Hepes, pH 7.6 (50 mM)
  
• 200µL of 0.5M EDTA (1 mM)
  
• 7 ml of 10% DOC (Na deoxycholate) (0.7%)
  
• 10 ml of 10% NP-40 (IPGEL) (1%)
  
• 10 ml of 5M LiCl or 2.12g powder (0.5 M)
  

Elution buffer

• 50mM Tris pH8
• 1mM EDTA
• 1% SDS

Procedure

IV. Chromatin Immunoprecipitation

1. Add 100 µL Ab prebound Dynal magnetic beads from step III.
2. Rock 4∞C 6 hrs to O/N.

Notes

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References

Relevant papers and books

1. Goldbeter A and Koshland DE Jr. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4. DOI:10.1073/pnas.78.11.6840 |
2. JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56. DOI:10.1016/s0022-2836(61)80072-7 |
3. ISBN:0879697164

Contact

• Who has experience with this protocol?