Light Orewood Brown Moon Fossil Af1

Cells were harvested and fixed in fresh Carnoy-water solution (100 g/L NaHCO~3~, 58 g/L acetic acid, 100 g/L D-glucose) for storage; cells were then re-suspended in ice-cold 100% EtOH and stored at −20 °C for further analysis. Cells were obtained using a Falcon isolation tube and centrifuged at 4 °C for 10 minutes at 8500 rpm. Pellet from cell suspension were thoroughly re-suspended in fresh Carnoy-water solution and the mixtures were loaded into a heat-resistant glass capillary (25 ml, 13 mm inner diameter, Thermo Scientific). The capillary was heated through a custom-designed stage at 60 °C for 2 h following which cells were collected and re-suspended in ice-cold 100% MeOH followed by centrifugation at 4 °C for 15 min at 3500 rpm to yield a cell pellet. Red blood cells were lysed by 1% Hanks buffer (140 mM NaCl, 5 mM EDTA, 2.4 mM KH~2~PO~4~, 3 mM EDTA, 85 mg/L NH~4~Cl, pH 7.4) and then filtered through two layers of glass wool. Following centrifugation at 4 °C for 5 min at 25,800 rpm, cell pellets were washed with water and then re-suspended in 100% MeOH. Cell number was measured with a Hemacytometer, DNA samples were stored at −80 °C until use. Spectrophotometry (Nanodrop 2000 or 260/280 nm) was used to measure concentration of DNA in cell suspensions. DNA samples were resuspended in 100 μl of TE buffer (Tris--HCl, pH 7.5, 1 M Tris--HCl, pH 8.0) and plated on MHA filters in duplicate (Costar) to detect genomic content. Proteins were extracted from frozen samples using 5 x SDS--Gel sample buffer (250 mM Tris--HCl, pH 6.8, 10% (v/v) glycerol, 0.