{mos_fb_discuss:5}
ES CELL/MOUSE TAIL GENOMIC DNA ISOLATION DSM
December 16, 20051. For ES cells: Grow cells to near-confluence in 24-well plates. Add 300 ul digestion buffer, with 1 mg proteinase K/ml, to each well and transfer to 1.5 ml microfuge tubes.
For tails: Place ~0.5-1 cm tail biopsy in 1.5 ml microfuge tube. Add 300 ul digestion buffer containing 1 mg proteinase K/ml.
2. Incubate "overnight" at 55∞C.
3. Add 150 ul saturated NaCl, and vortex vigorously x 15 sec. The solution will turn milky white.
[4. Tails only: Microcentrifuge, max rpm, RT, 5 min. Transfer sup to clean tube.]
5. Add 900 ul EtOH or 450 ul isopropanol. Solution should be clear except for DNA.
6. Microcentrifuge, max rpm, RT, 5 min. Aspirate. Wash pellet with 1 ml 70 % EtOH.
7. Carefully remove all EtOH, do not dry pellet, and immediately dissolve the pellet in 30 ul TE. Store at -20 C pending analysis.
8. Optional: Dilute 5 ul into 1 ml H2O and determine DNA concentration from A260/A280.
9. Digest 5 ul DNA solution (or 10 ug DNA if concentration was determined) with restriction enzyme for 4 hr, with repeat enzyme addition at 2 hr.
DNA solution 5 ul
10 X buffer 1.5 ul
restriction enzyme 1 ul
H2O 7.5 ul
total volume 15 ul
10. Separate digests on 1% agarose gel, blot to nylon membrane and hybridize to an appropriate probe.
Proteinase K 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
