1 M sorbitol - At room temperature.
1 M sorbitol - Chilled to 4°C.
Tris-EDTA (TE) - 10 mM Tris, pH 7.5; 1 mM EDTA.
β-ME buffer (11 ml) - 20 mM EDTA; 0.7 M β-ME. Prepare fresh at room temperature before each experiment.
Lyticase buffer (11 ml) - 1 M sorbitol; 50 mM Tris, pH 7.8; 5 mM β-ME. Prepare fresh at room temperature before each experiment.
Buffer A (4 ml) - 1 M sorbitol; 10 mM Tris, pH 7.5; 50 mM NaCl; 5 mM MgCl2; 1 mM CaCl2; 1 mM β-ME. Prepare fresh on ice for each experiment.
Buffer B (1.25 ml) - Buffer A with 0.15% NP-40. Prepare fresh on ice for each experiment.
Lyticase (from Arthrobacter luteus; catalog# L2524, Sigma) - Dilute powder to make a 20,000 U/ml stock in ddH2O. Store at -20°C.
Micrococcal nuclease (MNase) (catalog# N5386, Sigma) - Dilute powder to make a 2 U/μl stock in 10 mM HEPES, pH 7.5; 100 mM NaCl; 1 mM CaCl2; 50% glycerol. Store at -20°C.
MNase stop buffer (0.5 ml) - 5% SDS; 250 mM EDTA. Store at room temperature. Heat to 37°C or higher to dissolve SDS before using.
Proteinase K (0.5 ml) - 20 μg/μl in ddH2O. Store at -20°C.
RNase A (1 ml) - 0.1 μg/μl in TE. Store at -20°C.
Phenol, buffer saturated solution, pH 7.5-7.9
4 M NaCl
Ethanol (EtOH), 100%
Ethanol (EtOH), 70%
Eppendorf Phase Lock Gel Heavy, 2 ml (catalog# 0032-005.152, Eppendorf)
QIAquick Gel Extraction Kit (catalog# 28704, QIAGEN)
10x Taq buffer - 100 mM Tris, pH 8.3; 500 mM KCl; 20 mM MgCl2
dNTP mix - 8 mM total dNTPs (i.e., 2 mM each d(A|C|G|T)TP)
SYBR Green I (catalog# S-7567, Invitrogen) - Dilute 10,000x concentrate to a 100x stock using DMSO. Store protected from light at -20°C.
Q-PCR primer sets - Design primer pairs (e.g., using "Primer3":http://frodo.wi.mit.edu from the Whitehead Institute) to produce overlapping ==== 100 bp products tiling a particular region of the genome, centered every 30-50 bp, and with Tm~'s as close as possible to 60°C.
Yeast genomic DNA - Prepare from the same yeast strain where nucleosome positions will be assayed. Used for Q-PCR quantitation standards.