Isolation of genomic DNA from mammalian cells


Modified by Wenyi Feng in May 2015 from "Molecular Techniques and Methods" from Institute of Molecular Development LLC .

MATERIALS AND SOLUTIONS

Cell Resuspension Buffer (10 ml)
10 mM Tris-HCl pH 8.0      100 μl of 1M Tris-HCl pH 8.0
10 mM EDTA                        100 μl of 0.5 M EDTA pH 8.0
Add AGD H2O to a final volume of 10 ml, store at 4oC.

Proteinase K Solution
We have it in stock (10 mg/ml)

SDS solution
We have it in stock (20%) 

RNase A
We have it in stock (10 mg/ml) 

PROCEDURES

  1. Trypsinize, harvest and resuspend cells at 10E4 cells/μl (e.g., use 500 μl for 5 million cells) in Cell Resuspension Buffer. 
  2. To cell resuspension add SDS to a final concentration of 0.5% and Proteinase K to a final concentration of 200 μg/ml.
  3. Mix and incubate at  55oC for 2 hr.     
  4. Add 5 M NaCl to a final concentration of 0.2 M.
  5. Extract twice with equal volumes of phenol:chloroform:isoamyl alcohol (25:24:1) and once with chloroform.
  6. To evaporate the chloroform, leave tube open on the bench for 15 min.
  7. Add RNase A (DNase-free and protease-free) to a final concentration of 25 μg/ml and incubate for 1 hr at 37oC.  *I used 50 μg/ml.
  8. Extract once with phenol:chloroform:isoamy alcohol (25:24:1) and once with chloroform.
  9. Precipitate DNA by adding 1.5 volumes of 100% ethanol (stored at -20oC). 
  10. Centrifuge at 4oC for 15 min at 14,000 x rpm and removal liquid.
  11. Pulse-spin again to remove residual liquid with a yellow tip.
  12. Dry the pellet with tube open on the bench for 10 min.  
  13. Resuspend the pellet in TE pH 8.0.

1 comment:

  1. If isolating DNA from a sample containing 5 million cells resuspend the pellet in 200μl TE pH 8

    ReplyDelete