For CEN3, AluI is the diagnostic digest. Digest for several hours to overnight. Add RNase to bluejuice for Totals and Sups prior to loading onto gel. Run a 2.5% agarose gel. Transfer to GeneScreen. (I leave out the HCl treatment when preparing gels for transfer.). UV crosslink prior to hybridization.
Reagents Needed for ChIP
| 37% formaldehyde stock solution | 1 M DTT stock |
| 1 M TRIS, pH 9.4 stock | 1 mg/mL Oxalyticase stock or 10 mg/mL Zymolyase stock |
| PMSF, pepstatin A (1 mg/ml in 100% MeOH), leupeptin (1mg/ml in sterile water) stocks | |
| HEPES/sorb 20 mM HEPES, pH 7.4 1.2 M sorbitol |
250 mL: 5 mL 1 M HEPES 150 mL 2 M sorbitol |
| PIPES/sorb 20 mM PIPES, pH 6.8 1 mM MgCl2 1.2 M sorbitol |
250 mL: 5 mL 1 M PIPES 0.25 mL 1 M MgCl2 150 mL 2 M sorbitol |
| PBS | |
| Triton/HEPES Wash 0.25% Triton X-100 10 mM EDTA 0.5 mM EGTA 10 mM HEPES, pH 6.5 |
250 mL: 3.13 mL 20% Triton X-100 5 mL 0.5 M EDTA 0.5 mL 0.25 M EGTA 2.5 mL 1 M HEPES |
| NaCl/HEPES Wash 200 mM NaCl 1 mM EDTA 0.5 mM EGTA 10 mM HEPES, pH 6.5 |
250 mL: 10 mL 5 M NaCl 0.5 mL 0.5 M EDTA 0.5 mL 0.25 M EGTA 2.5 mL 1 M HEPES |
| SDS Lysis Buffer 1% SDS 10 mM EDTA 50 mM TRIS, pH 8.1 |
100 mL: 10 mL 10% SDS (UltraPure) 2 mL 0.5 M EDTA 5 mL 1 M TRIS |
| IP Dilution Buffer (for 1:9 dilution) 0.01% SDS 1.1% Triton X 100 1.2 mM EDTA 16.7 mM TRIS, pH 8.1 167 mM NaCL |
250 mL: 0.25 mL 10% SDS 13.8 mL 20% Triton X 100 0.6 mL 0.5 M EDTA 4.2 mL 1 M TRIS, 8.1 8.35 mL 5 M NaCl |
| Sonicated lambda DNA. Store at 4°C with 0.1% azide. | |
| Protein A Sepharose Bead Buffer: | 25 mL: 25 mL TE 25 mg BSA (Fraction V, powder) 25ul 10 % azide |
| TSE-150 Wash (= IP conditions) 0.1% SDS 1% Triton X-100 2 mM EDTA 20 mM TRIS-HCl, pH 8.1 150 mM NaCl |
250 mL: 2.5 mL 10% SDS 12.5 mL 20% Triton 1.0 mL 0.5 M EDTA 5.0 mL 1 M TRIS, 8.1 7.5 mL 5 M NaCl |
| TSE-500 Wash (Optional) 0.1% SDS 1% Triton X-100 2 mM EDTA 20 mM TRIS-HCl, pH 8.1 500 mM NaCl |
250 mL: 2.5 mL 10% SDS 12.5 mL 20% Triton 1.0 mL 0.5 M EDTA 5.0 mL 1 M TRIS, 8.1 25 mL 5 M NaCl |
| LiCl/Detergent Wash 0.25 M LiCl 1% NP-40 1% DOC 1 mM EDTA 10 mM TRIS-HCl, pH 8.1 |
250 mL: 2.65 g LiCl (42.39) 25 mL 10% NP-40 50 mL 5% DOC 0.5 mL 0.5 M EDTA 2.5 mL 1 M TRIS, 8.1 |
| TE, pH 8.0 | |
| Bead Elution Buffer 1% SDS/0.1 M NaHCO3 |
50 mL: 5 mL 10% SDS 5 mL 1M NaHCO3 |
| 5x Proteinase K Buffer 50 mM TRIS, pH 25 mM EDTA 1.25 % SDS |
10 mL: 0.5 mL 1 M TRIS 0.5 mL 0.5 M EDTA 1.25 mL 10 % SDS |
| Proteinase K Solution (Boehringer Mannheim, 1413 783), ~18.6 mg/mL | Glycogen stock |
