DNALaddering

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