At an extraction of DNA from a silica-dried plant leaves (50-60 mg), using a buffer consists of CTAB 2%, boric acid, Tris, EDTA, NaCl, Urea, and PVP, I got a small size pellet with low yield of DNA. I add 1 % Mercaptoethanol to the buffer at the time of the extraction and precipitate organic and non-organic substances with Phenol: Choroform-Isoamyl (25:24:1)for one time and with Choroform-Isoamyl (24:1) for two times. My plant is rich in polysaccharides which precipitated in the last supernatant with Ammonium Acetate. I would like to know, under these conditions, how can I increase the yield of DNA?
If you have difficult sample with all this troubles ...... it is best to try standard extraction kit to have good quality nucleic acid for further applications.
Dear Baeyen,
The buffer contains just 1 M urea as it is a chaotropic agent, and its role is obviously denature proteins.
Dear Mohamed Soltan,
Thanks a lot! Up to this moment, I didn't use commercial kits because AFLP requires highly pure DNA with a high quality and I thinks with Kits may get a good quantity of DNA but with many smears and fragmented DNA that is not good for the reaction.
Dear Mahamed,
I think, you need mild conditions for extraction. I usually perform lysis by buffer with ProtK (in place of urea) and without mercaptoethanol, and only one time Chloroform- Isoamyl (24:1).
The main loss DNA take place through stage Phenol: Chloroform-Isoamyl and Chloroform-Isoamyl. Sometimes enough reduce one replic to get 1.5-2 times more DNA.
Losses can be minimized by accuracy, especially in the precipitation step, when necessary gently and thoroughly mix the content of the tube.
Dear Mrs. Dr. Shapturenko,
I already add 10 or 20 mico-liter Prointase K (20mg/ml) after adding the buffer, then incubate the lysate at 60 degree for one hour.
As my plant leaves are rich in many compounds the most are polyphenols and polysaccharides, I got yellowish DNA in the first extraction when I used chloroform-Isoamyl two times. Also got gel-like DNA in the second extraction when used Phenol chloroform-Isoamy (25:24:1) for one time, then Ammonium Acetate, and finally chloroform-Isoamy before precipitating DNA with Isopropanol.
I didn't face this problem with the leaves of new seedlings of this plant. The problem is for the trees which I'm studying.
If you have polyphenols in your samples then it will surely make a difference in getting a good amount of DNA. 5-10mM Thiourea in extraction buffer will be helpful to inhibit polyphenol oxidases in your sample, and will also increase DNA yield. Hope this helps. Good Luck
I had similar issues with biofilm extractions using Phenol/Chloroform methods due to polysaccharides mostly from cyanobacteria, and humic remnants which contained polyphenols.
You can use 1.25-1.5M NaCl in a 50:50 mix of H2O & diethyl ether to remove the PS and polyvinyl pyrrolidone in initial processing of the leaves to remove the polyphenols. It's best to be gentle with initial mechanical processing of the leaves and performed at coldest possible temperature. You can also avoid Phenol with a CTAB method. There are some good commercial kits that are designed to deal with these contaminants and produce very high quality DNA also.
You can precipitate your DNA with 2-Propanol at -20 for 30 to 45 min.
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