a problem of genomic DNA purification - (May/09/2006 )

Hi,

I'm a new comer here, and hope we can have a nice discussion in the future.

Recently I met a problem when I try to purify genomic DNA, and it has bothered me a lot.

I follow the protocol from a lab in NIH,

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°C (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 °C (1200
rpm). 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°C.

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

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°C.

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


Everything seems all right until I add the EtOH to precipitate the DNA.
There will be something like organic solvent and my DNA at the bottom of the tube, and I can't spool out the DNA using glass pipette.

I tried many times but they all looked the same.
Does anyone have any idea about this?

M.Y.

just to be sure, for DNA precipitation, did you use isopropanol? or ethanol? with addition of sodium acetate you would use 100% ethanol, 3X vol isopropanol seems rather excessive, if anything I use 0.7X vol isopropanol and no sodium acetate to precipitate my gDNA with no problems, I think the organic layewr would be your isopropanol.

Nick

I agree that the isopropanol volume seems high, but I think the organic portion is likely carryover from your phenol:chloroform extraction. When transfering the aqueous layer, I'd rather leave some behind than try to get every last bit and carry phenol along...