episome - episome isolation (May/18/2006 )
does anyone has a protocole to isolate dna from an episome from tissue.
in short I want to get ride of the DNAfrom the nulceus and just get the episome.
[u]Low molecular weght DNA isolation procedure[u]
First, resuspend 10^6 cells (containing episome) in 1 ml of a mixture of:
- 10 mM Tris-HCl (pH 8.0),
- 10 mM EDTA,
- and 1 mg of pronase/ml.
Incubated at 37°C for 2 hours. Add 0.25 ml of 5 M NaCl and incubation overnight at 4°C.
Spin at 15,000 × g for 30 min, remove the pecipitant (genomic DNA), transfer supernatant to fresh tube. The supernatant was phenol-chloroform extracted; ethanol precipitated; resuspended in a mixture of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 25 µg of DNase-free RNase (Boehringer Mannheim)/ml; and incubated at 37°C for 30 min.