58
HANS GERD NOTHW ANG AND FRIEDHELM HILDEBRANDT
Procedure
ln advance
1. In advance prepare the following solutions on ice:
Extraction buffer:
Component
Stock
Final
Amount
Concentration
Concentration
a. Tris-HCl (pH 8.0) 1M
lOmM
O.Sml
b. EDTA (pH 8.0)
0.5 M
lOOmM
10 ml
c. RNAse Aa
10 mg/ml
20 )..lg/ml
0.1 ml
d. SDS
10%
0.5 o/o
2.5 ml
e. H20
36.9 ml
a DNAse free. To prepare RN Ase A free of DNAse, dissolve RN Ase A in TE-buffer at
10mglml, and heat between 75-100 oc for 15 min. Allow to cool down slowly. Store
aliquots at -20 oc.
If using blood samples, prepare the following anticoagulant acid citrate
dextrose solution (ACD):
Component
Amount
a. citric acid
0.48 g
b. sodium citrate
1.32 g
c. glucose
1.47 g
d.H20
to 100 ml
DNA isolation
2a) Cells growing in monolayers: Wash the monolayers twice with ice-cold
TBS. Add 0.5 ml TBS and scrape cells. Transfer cell suspension to a microcentrifuge tube, stored on ice. Wash the Petri dish with 1ml ofTBS
and add the washing to the centrifuge tube. Spin at 1500 g for 10 min,
4°C. Preheat water bath to 37 oc. Wash the cells in 5-10 volumes oficecold TBS and repeat centrifugation. Resuspend cells in TE-buffer (pH
8.0) at a concentration of 5 x 10 7 cells/ml. Transfer the solution to an
Erlenmeyer flask (for n ml of cell suspension, use a 50 x n ml flask). Add
1 Oml of extraction buffer for each milliliter of cell suspension. Break
pellet to 2-3 pieces with a pipet. Incubate the solution for 1h at 37°C
in a water bath, and then proceed to step 3.
HANS GERD NOTHW ANG AND FRIEDHELM HILDEBRANDT
Procedure
ln advance
1. In advance prepare the following solutions on ice:
Extraction buffer:
Component
Stock
Final
Amount
Concentration
Concentration
a. Tris-HCl (pH 8.0) 1M
lOmM
O.Sml
b. EDTA (pH 8.0)
0.5 M
lOOmM
10 ml
c. RNAse Aa
10 mg/ml
20 )..lg/ml
0.1 ml
d. SDS
10%
0.5 o/o
2.5 ml
e. H20
36.9 ml
a DNAse free. To prepare RN Ase A free of DNAse, dissolve RN Ase A in TE-buffer at
10mglml, and heat between 75-100 oc for 15 min. Allow to cool down slowly. Store
aliquots at -20 oc.
If using blood samples, prepare the following anticoagulant acid citrate
dextrose solution (ACD):
Component
Amount
a. citric acid
0.48 g
b. sodium citrate
1.32 g
c. glucose
1.47 g
d.H20
to 100 ml
DNA isolation
2a) Cells growing in monolayers: Wash the monolayers twice with ice-cold
TBS. Add 0.5 ml TBS and scrape cells. Transfer cell suspension to a microcentrifuge tube, stored on ice. Wash the Petri dish with 1ml ofTBS
and add the washing to the centrifuge tube. Spin at 1500 g for 10 min,
4°C. Preheat water bath to 37 oc. Wash the cells in 5-10 volumes oficecold TBS and repeat centrifugation. Resuspend cells in TE-buffer (pH
8.0) at a concentration of 5 x 10 7 cells/ml. Transfer the solution to an
Erlenmeyer flask (for n ml of cell suspension, use a 50 x n ml flask). Add
1 Oml of extraction buffer for each milliliter of cell suspension. Break
pellet to 2-3 pieces with a pipet. Incubate the solution for 1h at 37°C
in a water bath, and then proceed to step 3.