ProtocolforIsolationofGenomicDNAfromMammalianCells

Protocol for Isolation of Genomic DNA from Mammalian Cells

1.Trypsinize, harvest and resuspend cells at 107 / ml in 10 mM Tris-HCl (pH 8.0), 10 mM EDTA.

2.Add SDS and Proteinase K to a final concentration of 0.5% and 200 µg/ml, respectively.

3.Mix and incubate at 55°C for 2 hours.

4.Add NaCl to a final concentration of 0.2M.

5.Extract twice with equal volumes of phenol:chloroform (1:1) and once with chloroform. Phenol must be equilibrated with buffer before use.

6.Place the tube, with cap open, in 55°C water bath for 1 hour, to evaporate the chloroform.

7.Add Ribonuclease A (RNase A), DNase-free to a final concentration of 25 µg/ml and incubate for 1 hour at 37°C. The concentration of the enzyme may vary for different cell types.

8.Extract once with phenol:chloroform (1:1) and once with chloroform. Precipitate DNA with 1.5 volumes of ethanol.

9.Centrifuge at 10000x g for 5 minutes to pellet the DNA .

10Resuspend the pellet in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA.

References

1.Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1.31-1.38, 2001.

2.Sharma, R.C., et al., A rapid procedure for isolation of RNA-free genomic DNA from mammalian cells, BioTechniques, 14, 176-178, 1993.