Sperm Nuclei Preparation

1. 1X Nuclear Preparation Butter (NPB): 250 mM sucrose (1.5 M stock; filter-sterilize and store aliquots at -20°C), 15 mM HEPES (1M stock; titrate with KOH so that pH 7.7 is at 15 mM, filter-sterilize, and store aliquots at -20°C) (see Note 4), 0.5 mM spermidine trihydrochloride (Sigma S-2501; 10 mM stock; filter-sterilize and store aliquots at -20°C), 0.2 mM spermine tetrahydrochloride (Sigma S-1141; 10 mM stock; filter-sterilize and store aliquots at -20°C), 1 mM dithiothreitol (Sigma D-0632; 100 mM stock; filter-sterilize and store aliquots at -20°C) (see Notes 4 and 5).

For steps requiring protease inhibitors, add: 10 |g/mL leupeptin (Boeringer Mannheim 1 017 101; 10 mg/mL stock in DMSO, store aliquots at -20°C), 0.3 mM phenylmethylsulfonyl fluoride (PMSF) (Boeringer Mannheim 837 091; 0.3 M stock in EtOH, stored at -20°C).

2. 1X Marc's Modified Ringer (MMR): 100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM HEPES, pH 7.5. Prepare a 10X stock, and adjust pH with NaOH to 7.5. Sterilize 10X and 1X solutions by autoclaving.

3. Lysolecithin: 100 ||L of 10 mg/mL l-a-lysophosphatidylcholine (Sigma Type I, L-4129); dissolve at room temperature just before use. Store solid stock at -20°C.

4. Bovine serum albumin (BSA) (store at -20°C in 1 mL aliquots): 10% (w/v) BSA (fraction V, Sigma A-7906; prepare stock in water, titrate to pH 7.6 with KOH).

5. Sperm dilution buffer (store at -20°C in 0.5-mL aliquots): 250 mM sucrose, 75 mM KCl, 1 mM EDTA (0.5 M stock, pH8), 0.5 mM spermidine trihydrochloride (Sigma S-2501; 10 mM stock; filter-sterilize and store aliquots at -20°C), 0.2 mM spermine tetrahydrochloride (Sigma S-1141; 10 mM stock; filter-sterilize and store aliquots at -20°C), 1 mM dithiothreitol (Sigma D-0632; 100 mM stock; filter-sterilize and store aliquots at -20°C).

6. Hoechst No. 33342 (Sigma B-2261): 10 mg/mL stock in dH2O; store in a light-tight vessel at -20°C.

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