DNA Laddering

I. Protocol 1. Harvest cells Optional: wash plate with 37°C PBS (gently, so as not to lose cells); check on microscope, after aspirate PBS or media Place plate on ice Lyse with DNA ladder buffer 4°C 150 ul/60 mm plate Scrape, transfer to eppendorf Rotate 4°C 20 min 2. Microcentrifuge 15 min, 4°C To pellet chromatin Transfer sup 3. Phenol/CHCl3 extract Add equal volume phenol/CHCl3 Mix by pipetting Microfuge 2 min; transfer sup Will often get a very large interface 4. Optional: re-extract organic Add 300-500 ul TE8.0; mix; spin; pool sups 5. Ethanol Precipitate In eppendorf tube or 15 ml tube, depending on total sup volume Add 1/10 vol. 3 M NaOAcetate, 2 vol. ethOH �20°C overnight 6. Recover DNA Centifuge EtOH ppt: If in: 15 ml tubes: Sorvall SA600, 8,000 RPM, 10 min, 4°C epp tubes: microfuge 15 min, 4°C If was in 15 ml tube, probably best to resuspend in small volume (e.g., 500 ul TE), then re- EtOH ppt in epp tube, so pellet will be tight 80% EtOH wash: add ≈500 ul; vortex; re-spin Speed-vac dry Resuspend in TE 8.0: ≈25 ul/eppendorf; room temp ≈30 min 7. RNase Add 1ul 1 ug/ul RNase A; room temp 30 min 8. Gel electrophoresis Add 5X loading buffer (*omit bromophenol blue: can interfere with band visibility; may be OK to add xylene cyanol) Run on 1.2% agarose gel, in TAE buffer II. Reagents DNA Laddering Buffer: 40 ml 0.5% Triton X-100 800 ul 25% 5 mM Tris pH 7.4 200 ul 1M 20 mM EDTA 1.6 ml 500 mM 37.4 ml dH2O III. Notes Reference: Hockenberry et al. Nature