DNA Preparation from Adherent Cells

Section of Cancer Genomics,Genetics Branch,NCI

National Institutes of Health

Reagents

Chloroform

Mallinckrodt,cat.4440

EDTA,0.5 M

Ethanol,100%

Ethanol,70%

Isoamyl Alcohol

Sigma,cat.I-3643

Phenol

Gibco BRL,Cat.15513-039

Phosphate Buffered Saline (PBS),10X and 1X

Gibco BRL,Cat.10010-023

Proteinase K

Gibco BRL,Cat.24568-2

Sodium acetate,3 M,pH 5.2

Sodium dodecyl sulfate (SDS),10%

Tris-HCl,1 M,pH 8.0

TAE buffer (Tris acetate/disodium EDTA),1X

Bio Whittaker,Cat.16-011V

Trypsin

Gibco BRL,Cat.25200-056

Distilled Water

Gibco BRL,Cat.15230-170

Preparation

DNA buffer

1M Tris-HCl,1 M,pH 8.0 100 ml

0.5 M EDTA 100 ml

dH₂O water 300 ml

Chloroform/Isoamyl alcohol 24:1

Chloroform 24 ml

Isoamyl alcohol 1 ml

Procedure

1.Use trypsin or cell scraper to remove cells from tissue culture flask (T-75).Centrifuge cultured cells for 10 min at 10℃ (1200 rpm).Remove supernatant and re-suspend cell pellet in 1X PBS and wash twice with 10 ml 1X PBS,centrifuging between washes.

2.Resuspend pellet in 10 ml DNA buffer.Centrifuge cells for 10 min at 10 ℃ (1200rpm).Remove supernatant.

3.Add 3 ml DNA-buffer,re-suspend the pellet,add 125 ml Proteinase K (10 mg/ml) and 400 ml 10% SDS; shake gently and incubate overnight at 45℃.

4.Add 3.6 ml of phenol,shake by hand for 10 minutes (RT); centrifuge for 10 min at 10℃ (3000 rpm).

5.Transfer the supernatant into a new tube (15 ml); measure the volume.Add 1.8 ml phenol and 1.8 ml chloroform/isoamylalcohol (24:1)or a total amount equal to the volume of the supernatant.Shake by hand for 10 min (RT); centrifuge for 10 min at 10℃ (3000 rpm).

6.Transfer the supernatant into a new tube (15 ml); measure the volume.Add 3.6 ml chloroform/isoamylalcohol (24:1)or an amount equal to the volume of the supernatant.Shake by hand for10 min (RT); centrifuge for 10 min at 10℃ (3000rpm).

7.Transfer the supernatant into new tube,measure the volume.Add 1/10 volume 3 M sodium acetate (pH 5.2)and 3 x the volume 100% isopropanol (2-propanol);shake gently until the DNA is precipitated.

8.Use a sterile glass pipette to transfer the precipitated DNA into a tube with 30 ml of 70% ethanol tube.Place on inverting rack and invert for 2 hr to thoroughly rinse.Transfer DNA into a sterile eppendorf tube.

9.Centrifuge for 20 min at 14,000 rpm.Dry pellet in a SpeedVac for 5 min.Dissolve the DNA in 300-500 μl sterile water and place in an eppendorf thermomixer shaker overnight at 37℃.

10.Measure the DNA concentration and run 1-5 μl (approximately 200 ng)for gel electrophoresis on agarose gel (1%)in 1X TAE buffer.