Top > ŽΐŒ±ƒvƒƒgƒR[ƒ‹ > Materials & Methods > List > Genomic DNA isolation
œœœœœ@Genomic DNA isolation@œœœœœ



  1. Materials

    1. STE buffer pH 9.0
      10mM Tris-HCl
      100 mM NaCl
      1 mM EDTA
      pH the solution to 9.0 with NaOH

    2. STE saturated Phenol
      1. Melt the solid phenol in a 50 degree water bath.
      2. Add 20% of the above STE Buffer (v/w).
      3. Shake until the two become an emulsion.
      4. Allow the phases to separate.
      5. Decant off the aqueous phase.
      6. Add Hydroxyquinolin to 0.1% (w/v) and Beta-mercaptoethanol (BME) to 0.2% (v/v).
      7. Add the same amount of STE as you did in step 2) and store at 4 degrees.
        ( It may take more than one time of mixing to an emulsion to saturate the phenol)

    3. 50mM Tris-HCl, 0.1 mM EDTA

    4. 30% 4-Aminosalicylate,5% SDS (10 ml)
      3 g 4-Aminosalicylate
      5 ml 10% SDS

  2. Methods

    1. Grow 500 ml of cells to late log phase in appropriate medium (this will yield 2-3 g of E. coli in a rich medium).
    2. Spin cells down in 250 ml centrifuge bottles(7.5K in GSA rotor; 20 min.) You will want a well-packed pellet.
    3. Drain the pellet well and wipe the sides of the bottle with a Kimwipe.
    4. Resuspend 1 gram of cells in 15 ml high pH STE in a screw cap bottle.(Many plastics are trashed with phenol and chloroform, Nalgene and polypropylene are OK, but polycarbonate is not.)
    5. Add 2 ml 10 mg/ml lysozyme in H2O (make this fresh each time), incubate at 37‘C for 5 min. (the suspension should become less turbid).
    6. Add 3ml 30% 4-aminosalicylate, 5% SDS; and gently mix for 1 min.
    7. Hold at 70‘C for 10 min. The suspension should become completely clear.
    8. Transfer to a 50 ml dispo centrifuge tube containing 20 ml phenol saturated with high pH STE buffer and put on the octopus for 30 min. at room temperature. (You may need to add more STE to maintain sample volume.)
    9. Spin the tube in a clinical centrifuge at top speed for 10 min.
    10. With a wide mouthed 25 ml pipette, remove the aqueous phase to a fresh tube containing 25 ml phenol. (Genomic DNA is very susceptible to shear damage.)
    11. Repeat the extraction and phase separation 2 times.
    12. Transfer the aqueous phase to a 40ml Nalgene Oak Ridge tube and extract with 20 ml of chloroform for 10 min. on the octopus. Spin 5 min. in clinical centrifuge.
    13. Bring the aqueous to 20 ml with high pH STE and add 1 ml of RNase A* at 50 mg/ml (the RNase solution should be heated to 95‘C for 10 min. prior to use).*Use only disposable labware when working with RNase A
    14. Transfer to a dialysis bag and dialize for 2-3 hours against 500 ml high pH STE buffer at room temperature. (Be sure to check bag for leaks prior to use.) This will allow the ribonucleotides to escape the bag.
    15. Transfer to a dispo tube and add proteinase K to 50 ug/ml and SDS to 0.5%. ( 1.25 mg protenase and 1.25ml 10% SDS for 25 ml ).
    16. Incubate for 1 hour at 50‘C.
    17. Extract with 20 ml of high pH STE saturated phenol; followed by 20 ml of 1:1 phenol : chloroform ; and finally with 10 ml of chloroform.
    18. Split the aqueuos phase into two corex tubes and add 1ml 3.0 M NaOAc, and 20 ml absolute ethanol to each tube. Chill in the -20 freezer for at least 1 hour.
    19. Spool the DNA out of the tube with a Pasture pipette and transfer the DNA to an Oak Ridge tube containing 2-3 ml/(gram of original cells) of 50 mM Tris-HCl, 0.1mM EDTA (pH 8.0). Use the octopus at a slow setting or a slow reciprocal shaker to resuspend the DNA. Do not vortex. It may take several hours to a few days to resuspend the DNA.
    20. Determine the concentration of the DNA by UV absorbance (A260 and A280). It is a good idea to try a restriction digest on the DNA and run it out on an agarose gel. Make sure that the DNA cuts well and that there is little RNA contamination.
    21. Store at 4‘C. For long term storage, add a drop of CHCl3 to retard any bacterial and/or fungal growth. The chloroform can be evaporated from samples by a brief incubation at 37‘C.


Pacelabs

Top > ŽΐŒ±ƒvƒƒgƒR[ƒ‹ > Materials & Methods > List > Genomic DNA isolation