spacer
spacer
spacer
line.small
spacer.gif
 

Resources

How to isolate gnomic DNA from mES

1
When the media in the 24-well plates is yellow it’s time to harvest the cells.
2
Flick the media out of the plates and blot dry a little.
3
Add 0.5ml of Lysis buffer with proteinase K in it at 0.1mg/ml.
4
Seal each plate in a heat-sealing bag and place at 60 C overnight.
5
The next morning (it’s even OK to let it go 2 days!) remove the plates and let cool at room temp, then add 0.5ml of isopropanol and mix on a rotator for 20 min., you will see a white DNA precipitate.  It is now OK to store the plates at 4 deg C for a few days, or go to step 6.
6
Pick the DNA out of the wells, leaving as much of the isopropanol behind as possible, and put DNA into 100µl of TE.
7
Dissolve overnight at 60 o C, then store at 4 C
 
Lysis Buffer
Reagent
F
diln
per liter
Tris-HCl pH8, 2M
0.1M
5:100
50ml
EDTA 500mM
5mM
1:100
10ml
SDS 20%
0.2%
1:100
10ml
NaCl 5M
0.2M
4:100
11.69g
Prot K  20mg/ml
0.1mg/ml
1:200 or 5µl per ml
added fresh