help with DNA extraction

[Deanne Bell]

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.

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Deanne Bell
USDA Agricultural Research Service
9611 South Riverbend Avenue
Parlier, CA 93648
(559) 596-2806  voice
(559) 596-2791  fax
dbell at fresno.ars.usda.gov
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:-----Original Message-----
:From: bev at tissugen.com.au [mailto:bev at tissugen.com.au]
: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?
:
:Thanks, 
:Bev.
:
:
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