PPDI + DAPI solution

Recipe

Reagent stock	Amount to add (for 100 mL)	Final concentration
CaCl₂ (2.5 M, autoclaved)	80 μL	2 mM
EGTA (0.5 M, pH 8.0; autoclaved)	200 μL	1 mM
Glycerol (autoclaved)	78 mL	78%
HEPES-KOH (0.5 M, pH 7.8) (Note: Filter before use.)	1 mL	5 mM
KCl (4 M, autoclaved)	0.5 mL	20 mM
MgSO₄ (1 M, autoclaved)	1 mL	10 mM
NaCl (5 M, autoclaved)	2 mL	100 mM
Phenylenediamine (p-phenylenediamine; Sigma-Aldrich)	100 mg	1 mg/mL

Aliquot into 495-μL portions. Store for up to 1 mo at -80°C. (Note: Aliquots that turn brownish in color should be discarded and a fresh batch prepared.)
Before use, thaw an aliquot protected from light and add 5 μL of:
DAPI (4′,6-diamidine-2′-phenylindole dihydrochloride) (5 μg/mL; Sigma)		50 ng/mL