Spin for 4 min at max Note: if aqueous phase is cloudy, extract again. Transfer aqueous layer to a new 1.7 mL Eppendorf tube. Add 16 µL of 5 M NaCl (final conc. = 200 mM) and 2 µL of 20 mg/mL glycogen (40 µg total) to each sample and vortex or pipet up and down to mix. Add 920 µL cold 100% EtOH and vortex briefly. Incubate at -80°C for 30 min or until frozen solid. Spin at 20,000 x g for 15 min at 4°C. Wash pellet with 1 mL cold 70% EtOH and spin for 5min at 4°C at 20,000 x g. Remove EtOH using a pipet without disturbing the DNA pellet. Dry the pellet for 5 min at room temperature. Thoroughly resuspend the pellet in 50 10 mM Tris. μL Measure DNA concentration (e.g. using NanoDrop) and check the size of the fragmentation on an agarose gel. Average size of chromatin fragmentation should be 200bp. Store the remaining material at -20°C until needed. It can be prepared for sequencing and used as an input-control. Reagent Stock Concentration Final Concentration Volume for 50 mL Tris, pH 8.0 1 M 10mM 0.5 mL EDTA 0.5 M 1mM 0.1 mL SDS 10% 1% 5 mL dH2O -- -- 44.4 mL
Spin for 4 min at max Note: if aqueous phase is cloudy, extract again. Transfer aqueous layer to a new 1.7 mL Eppendorf tube. Add 16 µL of 5 M NaCl (final conc. = 200 mM) and 2 µL of 20 mg/mL glycogen (40 µg total) to each sample and vortex or pipet up and down to mix. Add 920 µL cold 100% EtOH and vortex briefly. Incubate at -80°C for 30 min or until frozen solid. Spin at 20,000 x g for 15 min at 4°C. Wash pellet with 1 mL cold 70% EtOH and spin for 5min at 4°C at 20,000 x g. Remove EtOH using a pipet without disturbing the DNA pellet. Dry the pellet for 5 min at room temperature. Thoroughly resuspend the pellet in 50 μL 10 mM Tris. Measure DNA concentration (e.g. using NanoDrop) and check the size of the fragmentation on an agarose gel. Average size of chromatin fragmentation should be 200bp. Store the remaining material at -20°C until needed. It can be prepared for sequencing and used as an input-control. Reagent Stock Concentration Final Concentration Volume for 50 mL Tris, pH 8.0 1 M 10mM 0.5 mL EDTA 0.5 M 1mM 0.1 mL SDS 10% 1% 5 mL dH2O -- -- 44.4 mL
Ren%20Lab%20Tissue%20Fixation%20and%20Sonication%20Protocol.docx