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ES CELL RAPID SCREEN: GENOMIC DNA ISOLATION DSM
Ref: Laird et al.(1991)19(15)4293December 16, 2005
1. Grow ES cells in 24-well plates.
2. As each well nears confluence, replace the medium with 300 ul digestion buffer containing 1 mg proteinase K/ml and return to humidified 37 ∞C, CO2 incubator. Continue until all samples have been incubated for at least several hours in proteinase K/digestion buffer.
3. Add 150 ul saturated NaCl. Either vortex the entire 24 well plate on a plate vortex platform (easier), or mix thoroughly with a Pasteur pipet (better). The solution will turn milky white. Shearing of genomic DNA at this step is desireable because it facilitates dissolution of the final ppt in TE.
4. Add 900 ul EtOH or 450 ul isopropanol and mix thouroughly. Solution should be clear except for DNA.
5. Transfer the DNA ppt to 30-100 ul TE in a "V" microtiter well using P-200 tip.
6. Optional: Dilute 5 ul into 1 ml H2O and determine DNA concentration from A260/A280.
7. Digest 10 ug DNA (or 5-15 ul if DNA concentration was not determined) with restriction enzyme, separate on 1% agarose gel, blot to nylon membrane and hybridize to an appropriate probe.
Digestion Buffer:
Stock
for 1 liter
for 0.5 liter
20 mM Tris pH 8.0
1 M
20 ml
10 ml
10 mM NaCL
5 M
2 ml
1 ml
10 mM EDTA
0.5 M
20 ml
10 ml
0.5% SDS
20%
25 ml
12.5 ml
H2O
---
to 1000 ml
to 500 ml
