60
HANS GERD NOTHWANG AND FRIEDHELM HILDEBRANDT
5. Spin at 400 g for 15 min, RT.
6. Transfer the viscous aqueous phase with a wide-bore pipet (3 mm-diameter orifice) to a clean centrifuge tube and repeat steps 4-5 twice.
7. a) To isolate very-high-molecular-weight DNA (>200 kb), dialyze the
aqueous phase at 4 oc four times against 41 of a solution of 50 mM
Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0) until the OD 270 ofthe dialysate is less than 0.05. Allow room in the dialysis bag for the volume
of the sample to increase 1.5 to 2-fold. Continue with step 15.
b) To isolate smaller sized DNA add l/12th of the volume of 5 M Na Cl,
mix by slowly turning the tube. Add 2.5 vol of ice-cold 100% ethanol.
8. Keep at 4 oc for 10 min.
9. Spin at 5000 g for 10 min.
10. Remove the ethanol carefully.
11. Add 50 ml 70% ethanol to remove precipitated salt, mix briefly.
12. Repeat step 9.
13. Store the pellet in an open tube at room temperature until the last visible
traces of ethanol have evaporated.
Note: Do not allow the pellet to dry completely; otherwise, it will be very
difficult to dissolve.
14. Add 1ml ofTE-buffer (pH 8.0) for each 5 x 10 6 cells. Place the tube on a
rocking platform and gently rock the solution until the DNA has completely dissolved. This usually takes 12-24 h.
15. Measure the optical density of the DNA at 260 nm and 280 nm.
16. Expect 200 )lg from 5 x 10 7 cells or 20 ml of blood.
Subprotocol 3
Isolation of Cytoplasmic RNA
Principles of RNA isolation. The ability to isolate pure, intact RNA is essential in the study of gene expression, RT-PCR andin cDNA cloning. The
difficulty in isolating RNA is that ribonucleases are ubiquitous, very stable
and function under a variety of conditions unsuitable for most other enzymes. RNA is not shear-sensitive but it is very labile to alkali, so the pH
HANS GERD NOTHWANG AND FRIEDHELM HILDEBRANDT
5. Spin at 400 g for 15 min, RT.
6. Transfer the viscous aqueous phase with a wide-bore pipet (3 mm-diameter orifice) to a clean centrifuge tube and repeat steps 4-5 twice.
7. a) To isolate very-high-molecular-weight DNA (>200 kb), dialyze the
aqueous phase at 4 oc four times against 41 of a solution of 50 mM
Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0) until the OD 270 ofthe dialysate is less than 0.05. Allow room in the dialysis bag for the volume
of the sample to increase 1.5 to 2-fold. Continue with step 15.
b) To isolate smaller sized DNA add l/12th of the volume of 5 M Na Cl,
mix by slowly turning the tube. Add 2.5 vol of ice-cold 100% ethanol.
8. Keep at 4 oc for 10 min.
9. Spin at 5000 g for 10 min.
10. Remove the ethanol carefully.
11. Add 50 ml 70% ethanol to remove precipitated salt, mix briefly.
12. Repeat step 9.
13. Store the pellet in an open tube at room temperature until the last visible
traces of ethanol have evaporated.
Note: Do not allow the pellet to dry completely; otherwise, it will be very
difficult to dissolve.
14. Add 1ml ofTE-buffer (pH 8.0) for each 5 x 10 6 cells. Place the tube on a
rocking platform and gently rock the solution until the DNA has completely dissolved. This usually takes 12-24 h.
15. Measure the optical density of the DNA at 260 nm and 280 nm.
16. Expect 200 )lg from 5 x 10 7 cells or 20 ml of blood.
Subprotocol 3
Isolation of Cytoplasmic RNA
Principles of RNA isolation. The ability to isolate pure, intact RNA is essential in the study of gene expression, RT-PCR andin cDNA cloning. The
difficulty in isolating RNA is that ribonucleases are ubiquitous, very stable
and function under a variety of conditions unsuitable for most other enzymes. RNA is not shear-sensitive but it is very labile to alkali, so the pH