Cell Resuspension Buffer (1 liter)
10 mM Tris-HCl (pH8.0) ----------------------- 10 ml of 1 M Tris-HCl
10 mM EDTA ----------------------- 10 ml of 1 M Tris-HCl
Add deionized H2O to make a final volume of --- 1 liter
Keep at
4oC.
Proteinase K Solution (10 mg/ml)
Proteinase K ------------------------------------ 1 mg
Distilled H2O ------------------------------------ 1 ml
Store at
-20oC.
PROCEDURES
1. Trypsinize, harvest and resuspend cells at 107/ ml in Cell Resuspension Buffer.
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.
6. Place the tube, with cap open, in 55°C water bath for 1 hour, to evaporate the chloroform.
7. Add RNase A (DNase-free and protease-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.
9. Precipitate DNA with 1.5 volumes of ethanol.
10. Centrifuge at 10,000x g for 5 minutes to pellet the DNA.
11. Resuspend the pellet in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA.
NOTES
Do not over-dry the DNA pellet. It will be very difficult to dissolve the DNA in TE buffer.
KIT INFORMATION
REFERENCES
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.
Sharma, R.C., et al., A rapid procedure for isolation of RNA-free genomic DNA from mammalian cells, BioTechniques, 14, 176-178,
1993.