Total RNA was precipitated by adding 1/10 volume of 3 M sodium acetate pH 5.2 (NaOAcDEPC) and one volume of cold isopropanol.
This sample was then neutralised by adding 1/10th volume of 1 M Tris-HCl pH 8.8.
The subtracted RNA was precipitated by adding 1/10 volume of 3 M sodium acetate, and 2.5× volume of ethanol, and was maintained at −20°C for 30 minutes.
Cell lysis was obtained by adding 1/10 volume of glass beads and vortexing 6 cycles of 30 seconds spaced with 30 seconds cycles on ice.
For determination of LD content, aliquots were withdrawn during growth and cells were immediately fixed by adding 1/9 volume of formaldehyde (final concentration 3.7% v/v).
The reaction mixture was added in double the volume of the media and was kept in dark for 45 min and then the reaction was stopped by adding 1/10 volume of 1 N HCl to each well.
Sample acidification was done by adding 1/10 volume of 1 N HCl, incubating at room temperature for 10 min, and neutralizing with 1/10 volume of 1 N NaOH/0.1 M Tris.
Coagulation was started by adding 1 volume of buffer B (2.5 mM Z-GGR-AMC, 20 mM Hepes, 140 mM NaCl, 100 mM CaCl2 and 6% bovine serum albumin).
After chloroform∶octanol (24∶1) extraction, DNA was precipitated by adding 2/3 volume of isopropanol, and the pellet was washed in 70% ethanol; air-dried, and suspended in 400 µl of TE (500 mM Tris, 50 mM EDTA).
Urine samples were acidified to pH 3 by adding 1/10 volume of 1 N HCl.
