help with DNA extraction

Deanne Bell dbell at
Thu Aug 8 17:09:04 EST 2002

Hi Bev

I don't know what type of tissue you have been working on, I mostly work with plants - but I have a lot of experience with a wide variety of plants.  So I hope I can help.

Sometimes harvesting DNA at differenet times of year or at different times in the life cycle of your organism can make a difference in interferring substances.  It sounds to me like you may have some polysaccharides coming through in your preps.  In the past I have been able to clean preps up by doing several ETOHic ppts (10% vol. 2.5M Na acetate plus 250% vol ETOH; incubate at -20*C for 30 min; centrifuge, bring up pellet in 200ul TE and re-do this 2 - 3 times)

If you are really interested in just getting un-inhibitied PCRs, try decreasing the amount of template DNA you add (for example, try 0.5ul DNA prep in a 25ul reaction).  At some point the interfereing substance gets diluted enough that the PCR is no longer inhibited and it can usually amplify even small amounts of DNA.

Deanne Bell
USDA Agricultural Research Service
9611 South Riverbend Avenue
Parlier, CA 93648
(559) 596-2806  voice
(559) 596-2791  fax
dbell at

:-----Original Message-----
:From: bev at [mailto:bev at]
:For months now I have been doing DNA extractions from tissue 
:with no problems, getting good quality DNA suitable for PCR. 
:Following ProteinaseK/SDS digestion of the tissue, I carry out 
:an extraction with phenol/chloroform/isoamyl alcohol 
:mixture(purchased from ICN). I then precipitate with NaOAc and EtOH. 
:However, in the last few weeks I have run into problems. On 
:addition of the EtOH to precipitate the DNA, the solution 
:turns milky. This doesn't clear when I mix it, although I 
:still see DNA threads condensing. When I centrifuge the 
:solution, I get a DNA pellet down the bottom, along with a 
:viscous (sometimes brown, sometimes clear) droplet that I 
:think is phenol. The DNA will no longer PCR after this happens. 
:I have ordered new phenol/chloroform solution, this has not 
:helped. I have tried a straight chloroform extraction 
:following the phenol/chloroform solution, again this has not 
:helped. I have made up fresh extraction buffer and NaOAc 
:solution but still have the problem. 
:I am wondering, does this sound like a problem with the 
:phenol/chloroform solution? I note that the new bottle I got 
:is the same lot number as the last bottle, perhaps this is a 
:bad batch? Does anyone have any helpful hints?

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